CA2393556A1 - Transferrin assay - Google Patents
Transferrin assay Download PDFInfo
- Publication number
- CA2393556A1 CA2393556A1 CA002393556A CA2393556A CA2393556A1 CA 2393556 A1 CA2393556 A1 CA 2393556A1 CA 002393556 A CA002393556 A CA 002393556A CA 2393556 A CA2393556 A CA 2393556A CA 2393556 A1 CA2393556 A1 CA 2393556A1
- Authority
- CA
- Canada
- Prior art keywords
- transferrin
- content
- sample
- variants
- variant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 239000012581 transferrin Substances 0.000 title claims abstract description 225
- 102000004338 Transferrin Human genes 0.000 title claims abstract description 217
- 108090000901 Transferrin Proteins 0.000 title claims abstract description 217
- 238000003556 assay Methods 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 106
- 210000001124 body fluid Anatomy 0.000 claims abstract description 35
- 239000010839 body fluid Substances 0.000 claims abstract description 35
- 102000002070 Transferrins Human genes 0.000 claims abstract description 30
- 108010015865 Transferrins Proteins 0.000 claims abstract description 30
- 238000000926 separation method Methods 0.000 claims description 76
- 108010074568 disialotransferrin Proteins 0.000 claims description 51
- 108090001090 Lectins Proteins 0.000 claims description 47
- 102000004856 Lectins Human genes 0.000 claims description 47
- 239000002523 lectin Substances 0.000 claims description 47
- 239000003446 ligand Substances 0.000 claims description 47
- 210000002966 serum Anatomy 0.000 claims description 41
- 108010038196 saccharide-binding proteins Proteins 0.000 claims description 39
- 230000027455 binding Effects 0.000 claims description 31
- 102000001708 Protein Isoforms Human genes 0.000 claims description 24
- 108010029485 Protein Isoforms Proteins 0.000 claims description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 125000005629 sialic acid group Chemical group 0.000 claims description 7
- 210000004369 blood Anatomy 0.000 claims description 6
- 239000008280 blood Substances 0.000 claims description 6
- 101710150766 Sialic acid-binding lectin Proteins 0.000 claims 1
- 125000000837 carbohydrate group Chemical group 0.000 claims 1
- 208000007848 Alcoholism Diseases 0.000 abstract description 9
- 201000007930 alcohol dependence Diseases 0.000 abstract description 7
- 238000012544 monitoring process Methods 0.000 abstract description 5
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 108700022763 carbohydrate-deficient transferrin Proteins 0.000 description 52
- 239000000243 solution Substances 0.000 description 33
- 238000004128 high performance liquid chromatography Methods 0.000 description 29
- 150000001720 carbohydrates Chemical group 0.000 description 17
- 238000003127 radioimmunoassay Methods 0.000 description 17
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 16
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 12
- 235000014633 carbohydrates Nutrition 0.000 description 11
- 241000473945 Theria <moth genus> Species 0.000 description 10
- 238000005342 ion exchange Methods 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000007983 Tris buffer Substances 0.000 description 9
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 description 8
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 8
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 8
- 238000003018 immunoassay Methods 0.000 description 8
- 230000005291 magnetic effect Effects 0.000 description 8
- 239000002245 particle Substances 0.000 description 8
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 8
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 8
- 229920001213 Polysorbate 20 Polymers 0.000 description 7
- 102000023852 carbohydrate binding proteins Human genes 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 7
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 235000008995 european elder Nutrition 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 238000004255 ion exchange chromatography Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- 238000001556 precipitation Methods 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 238000011088 calibration curve Methods 0.000 description 5
- 108091008400 carbohydrate binding proteins Proteins 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 150000002482 oligosaccharides Chemical class 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 4
- 108010062580 Concanavalin A Proteins 0.000 description 4
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 4
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 4
- 235000004443 Ricinus communis Nutrition 0.000 description 4
- 240000000528 Ricinus communis Species 0.000 description 4
- 240000006028 Sambucus nigra Species 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 229910052742 iron Inorganic materials 0.000 description 4
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 229920001542 oligosaccharide Polymers 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- -1 sheets Substances 0.000 description 4
- 230000009870 specific binding Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000590002 Helicobacter pylori Species 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 3
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 3
- 108010017507 Ricinus communis agglutinin-1 Proteins 0.000 description 3
- 241000208829 Sambucus Species 0.000 description 3
- 235000003142 Sambucus nigra Nutrition 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229940037467 helicobacter pylori Drugs 0.000 description 3
- 238000001155 isoelectric focusing Methods 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 239000006249 magnetic particle Substances 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- 241000220457 Crotalaria Species 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 description 2
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 2
- 108010089814 Plant Lectins Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 235000018735 Sambucus canadensis Nutrition 0.000 description 2
- 108010046516 Wheat Germ Agglutinins Proteins 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 206010001584 alcohol abuse Diseases 0.000 description 2
- 208000025746 alcohol use disease Diseases 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- 238000005571 anion exchange chromatography Methods 0.000 description 2
- 108010009577 asialotransferrins Proteins 0.000 description 2
- 235000007123 blue elder Nutrition 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 235000007124 elderberry Nutrition 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 229930182830 galactose Natural products 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 150000004676 glycans Polymers 0.000 description 2
- 239000003456 ion exchange resin Substances 0.000 description 2
- 229920003303 ion-exchange polymer Polymers 0.000 description 2
- 239000004816 latex Substances 0.000 description 2
- 229920000126 latex Polymers 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000003726 plant lectin Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 150000004804 polysaccharides Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 101710186708 Agglutinin Proteins 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 101710141737 Bark lectin Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 108010067225 Cell Adhesion Molecules Proteins 0.000 description 1
- 102000016289 Cell Adhesion Molecules Human genes 0.000 description 1
- 101000945929 Crotalaria juncea Lectin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101000766307 Gallus gallus Ovotransferrin Proteins 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 239000012630 HPLC buffer Substances 0.000 description 1
- 101710146024 Horcolin Proteins 0.000 description 1
- 102000008133 Iron-Binding Proteins Human genes 0.000 description 1
- 108010035210 Iron-Binding Proteins Proteins 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- 101710189395 Lectin Proteins 0.000 description 1
- 241000239218 Limulus Species 0.000 description 1
- 241001521394 Maackia amurensis Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 101710179758 Mannose-specific lectin Proteins 0.000 description 1
- 101710150763 Mannose-specific lectin 1 Proteins 0.000 description 1
- 101710150745 Mannose-specific lectin 2 Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- JZLHQDUIAGSFDK-UHFFFAOYSA-K O.[Na+].[Na+].[Na+].N(CC(=O)[O-])(CC(=O)[O-])CC(=O)[O-].CC(C)O Chemical compound O.[Na+].[Na+].[Na+].N(CC(=O)[O-])(CC(=O)[O-])CC(=O)[O-].CC(C)O JZLHQDUIAGSFDK-UHFFFAOYSA-K 0.000 description 1
- SJEYSFABYSGQBG-UHFFFAOYSA-M Patent blue Chemical compound [Na+].C1=CC(N(CC)CC)=CC=C1C(C=1C(=CC(=CC=1)S([O-])(=O)=O)S([O-])(=O)=O)=C1C=CC(=[N+](CC)CC)C=C1 SJEYSFABYSGQBG-UHFFFAOYSA-M 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000000910 agglutinin Substances 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- BLFLLBZGZJTVJG-UHFFFAOYSA-N benzocaine Chemical compound CCOC(=O)C1=CC=C(N)C=C1 BLFLLBZGZJTVJG-UHFFFAOYSA-N 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 229940052299 calcium chloride dihydrate Drugs 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000012501 chromatography medium Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 229920003045 dextran sodium sulfate Polymers 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- KDQPSPMLNJTZAL-UHFFFAOYSA-L disodium hydrogenphosphate dihydrate Chemical compound O.O.[Na+].[Na+].OP([O-])([O-])=O KDQPSPMLNJTZAL-UHFFFAOYSA-L 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000002875 fluorescence polarization Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000000554 iris Anatomy 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000004848 nephelometry Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000009450 sialylation Effects 0.000 description 1
- BBMHARZCALWXSL-UHFFFAOYSA-M sodium dihydrogenphosphate monohydrate Chemical compound O.[Na+].OP(O)([O-])=O BBMHARZCALWXSL-UHFFFAOYSA-M 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/90—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving iron binding capacity of blood
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/79—Transferrins, e.g. lactoferrins, ovotransferrins
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/808—Automated or kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/811—Test for named disease, body condition or organ function
Abstract
The invention relates to an assay method for assessing a transferrin variant or combination of transferrin variants for the diagnosis and monitoring of alcoholism, and to kits for performing the assay. In particular, the invention provides a method of producing an algorithm for determining the content of a transferrin variant or combination of transferrin variants, preferably a CDT
variant or combination of CDT variants, in a sample of body fluid, said method comprising: (a) obtaining at least two solutions each having known contents of asialo- (A1, A2, A3, etc.) and disialo-transferrins (D1, D2, D3, etc.); (b) determining the content in each of said solutions of a transferrin variant or combination of transferrin variants fractions substantially free from tri- and higher sialylated transferrins; (c) determining the total transferrin variant content (T1, T2, T3, etc.) of said fractions; and (d) producing an algorithm capable of determining the content of any transferrin variant or combination of transferrin variants, preferably a CDT variant or combination of CDT
variants, in any given sample of body fluid subjected to said determination step b).
variant or combination of CDT variants, in a sample of body fluid, said method comprising: (a) obtaining at least two solutions each having known contents of asialo- (A1, A2, A3, etc.) and disialo-transferrins (D1, D2, D3, etc.); (b) determining the content in each of said solutions of a transferrin variant or combination of transferrin variants fractions substantially free from tri- and higher sialylated transferrins; (c) determining the total transferrin variant content (T1, T2, T3, etc.) of said fractions; and (d) producing an algorithm capable of determining the content of any transferrin variant or combination of transferrin variants, preferably a CDT variant or combination of CDT
variants, in any given sample of body fluid subjected to said determination step b).
Description
This invention relates to an assay method for assessing a transferrin variant or combination of transferrin variants for the diagnosis and monitoring of alcoholism, and to kits for performing the assay. In particular, the invention relates to an assay method for assessing a carbohydrate-deficient transferrin variant or any combination of such variants.
Serum transferrin is a glycoprotein with a molecular weight of about 80 kD which comprises a single polypeptide chain with two N-linked polysaccharide chains. These polysaccharide chains are branched and each chain may terminate in either two or three antennae, each with terminal sialic acid residues.
Wong and Regoeczi, in Int. J. Peptide Res. (1977) x:241-248, reported that human transferrin was naturally heterogeneous, occurring in variant forms with different levels of sialylation. Such variants are generally believed to be the hexasialo, pentasialo, tetrasialo, trisialo, disialo, monosialo and asialo transferrins.
Recently, it has been reported that the levels of monosialotransferrin are very low (see Landberg et al.
(1995) Biochem. Biophys. Res. Comm. 2,10(2):267-274 and Jochen et al. Biochemica et Biophysica Acta (1998) 1380:93-101).
The asialo, monosialo and disialo variants are referred to herein as carbohydrate-deficient transferrin or CDT. The asialo variant, now known to be completely devoid of carbohydrate chains, is referred to herein as carbohydrate free transferrin or CFT.
In the normal healthy individual, the tetrasialo variant appears to predominate; however it has been reported that the asialo, monosialo, disialo and, to some degree the trisialo variants occur in elevated levels in the blood of alcoholics (see van Eijk et al.
(1983) Clin. Chim. Acta ,;32:167-171, Stibler (1991) Clin. Chim. ,x:2029-2037 and Stibler et al. in "Carbohydrate-deficient transferrin (CDT) in serum as a marker of high alcohol consumption", Advances in the Biosciences, (Ed Nordmann et al), Pergamon, 1988, Vol.
71, pages 353-357).
CDT has been shown to be an effective marker for alcohol consumption, in particular for detecting and monitoring chronic alcohol consumption, and unlike conventional tests (e.g. quantitation of y-glutamyltransferase or measurement of mean corpuscular volume) can bemused to screen for heavy alcohol intake in patients with liver disease.
Recognition of the fact that the CDT profile of alcohol abusers differs from that of abstainers or normal users, combined with the identification of the relative amounts of each CDT isoform, e.g. on the basis of the differences in the isoelectric point (pI) or charge of the different transferrin molecules, has led to the development of several diagnostic assays for CDT.
These are described in the patent and scientific literature, for example in US-A-4626355, WO 91/19983, WO 96/26444, Heil et al. (1994) Anaesthetist 43:447-453, and Dumon et al. (1996) Clin. Biochem. x(6):549-553).
More recent studies (for example by Landberg et al.
(1995) Biochem. Biophys. Res. Comm. 210(2): 267-274), have shown, by releasing the N-glycans from each isoform of transferrin and analysing them by high-pH anion exchange chromatography, that the existence of disialo and asialo-transferrins correlates with the loss of one or both of the entire carbohydrate chains respectively from the transferrin polypeptide. This is confirmed by Jochen et al. in Biochemica et Biophysica Acta (1998) 1380:93-101.
Based on the discovery that the presence of transferrin isoforms which are completely devoid of carbohydrate (hereinafter referred to as carbohydrate free transferrin or CFT) is a strong indicator of WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 alcoholism in the absence of any knowledge of the prevalence of any other CDT variants (i.e. monosialo, disialo or trisialotransferrin variants), the Applicants developed and described in WO 99/00672 an assay for the determination of CFT as a means of assessing alcoholism.
The assay is robust, simple and quick to perform, and readily amenable to automation or compatible with existing routine clinical diagnostic laboratory procedures. This is achieved by separating the carbohydrate-containing transferrins from a sample by contacting the'sample with a carbohydrate-binding ligand, e.g. a lectin, and detecting and measuring the carbohydrate-free transferrin contained in the separated, non-binding fraction.
The accuracy and thus the clinical value of each of the assay procedures mentioned above is dependent on an efficient method of separation of the individual transferrin isoforms or combination of such isoforms.
As a result, relatively complex, expensive separation procedures are necessary to obtain an accurate diagnosis.
In particular, the pI or charge based methods of the prior art are primarily centred on procedures involving ion exchange chromatography. The difference in pI between the different transferrin variants is very narrow, down to 1/10 of a pH unit and therefore to effect separation of CDT variants, a very good separation is required. In the case of ion exchange chromatography, this constraint effectively means that a column format must be used; batch filtration-based ion exchange procedures do not provide a sufficient separation or resolution. Column formats are however less preferred in clinical chemistry or diagnostic procedures, due to their time consuming and labour intensive operation, problems of storage and transport, incompatibility with commonly-used systems etc.
Due to the low levels of CFT generally found in serum samples (even in alcoholics only about to of the transferrin molecules are asialotransferrin or CFT), an efficient separation is similarly required to effect separation of CFT from any carbohydrate containing transferrins in the assay described in WO 99/00672.
There is therefore a need for an alternative assay method in which incomplete or partial separation of transferrin variants, e.g. one or more CDT variants, can be tolerated without affecting the clinical value of the assay. The present invention seeks to address this need.
Martensson et al. (Alcoholism: Clin. and Exp.
Research (1997) x(9):1710-1715) describe a clinical study of transferrin variant concentrations by means of ion exchange HPLC in combination with RIA analysis of the transferrin content of the HPLC fractions eluted.
Higher clinical sensitivity was found for measurement of disialotransferrin alone and with the sum of asialo-, monosialo- and disialo-transferrins compared to asialo-and monosialo-transferrins alone. A much lower clinical sensitivity was found with trisialotransferrins.
M~rtensson et al. focus on the differences in clinical signals between the different transferrin variants with a view to selecting the best transferrin variant or combination of variants. No correlation figures for the different variants are reported.
In June 1999, Professor Jan-Olof Jeppson working at the University of Malmo, Sweden reported a weak correlation (correlation coefficient RZ - 0.63) between asialo and disialo-transferrins using the procedure of ion exchange chromatography and spectrophotometric detection at 470 nm as described in WO 95/04932.
Contrary to the findings of Jeppson, we have now surprisingly found a high level of correlation between the asialo- and disialo-transferrin contents of serum samples. This has led to the development of an improved assay procedure for determining the content of any desired transferrin variant or combination of transferrin variants, in particular asialo- or disialo-transferrin or CDT, in a body fluid sample in which a less efficient separation of transferrin variants can be tolerated. This allows much simpler separation methods to be used for the assessment of alcohol consumption.
Thus, according to one aspect, the present invention provides a method of producing an algorithm for determining the content of a transferrin variant or combination of transferrin variants, preferably a CDT
variant or combination of CDT variants, in a sample of body fluid, said method comprising:
(a) obtaining at least two solutions each having known contents of asialo- (A1, A2, A3, etc.) and disialo-transferrins (D1, D2, D3, etc.);
(b) subjecting each of said solutions to a separation method whereby to separate fractions substantially free from tri- and higher sialylated transferrins (c) determining the total transferrin variant content (T1, T2, T3, etc.) of said fractions; and id) producing an algorithm capable of determining the content of any transferrin variant or combination of transferrin variants, preferably a CDT variant or combination of CDT variants, in any given sample of body fluid subjected to said separation method.
As mentioned further below, however, the purpose of the separation step used in the methods herein described is essentially to remove all or substantially all tri-, tetra-, penta- and hexa-sialotransferrins (non-target variants) from the sample, allowing the remaining target variants (asialo-, monosialo- and disialotransferrins) to be determined Thus viewed from a broader aspect, the invention provides a method of producing an algorithm for WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 determining the content of a transferrin variant or combination of transferrin variants, preferably a CDT
variant or combination of CDT variants, in a sample of body fluid, said method comprising:
(a) obtaining at least two solutions each having known contents of asialo- (Al, A2, A3, etc.) and disialo-transferrins (D1, D2, D3, etc.);
(b) determining the content in each of said solutions of a transferrin variant or combination of transferrin variants in fractions substantially free from tri- and higher sialylated transferrins;
(c) determining the total transferrin variant content (T1, T2, T3, etc.) of said fractions; and (d) producing an algorithm capable of determining the content of any transferrin variant or combination of transferrin variants, preferably a CDT variant or combination of CDT variants, in any given sample of body fluid subjected to said determination step b) .
In general, the algorithm produced in accordance with the invention will be one capable of determining the actual content or amount of asialo- or disialo-transferrin, or the actual content or amount of asialo-, monosialo- and disialo-transferrins (i.e. CDT) in any sample of body fluid.
As will be apparent, the algorithm will be specific to a given determination step or separation method carried out under specific conditions. Preferably, the algorithm for use in the invention will be based upon a quantitation of each of the isoforms of transferrin having zero, one or two sialic acid residues per molecule. Most preferably, the algorithm may be produced on the basis of the high correlation between -the content of asialo- (CFT) and disialo-transferrins in a sample, preferably also the low level of monosialotransferrin.
Based on the correlation determined between asialo-and disialotransferrins, the invention essentially provides a method of producing a calibration curve specific to any given determination step or separation method and which may be used to calculate the content or amount of any transferrin variant or variants in any given sample of body fluid. The correlation coefficient between asialo,'- and disialotransferrin may be determined using mathematical techniques and correlations standard in the art, e.g. least squares analysis.
More specifically, the algorithm produced in accordance with the invention may be defined by at least one of the following equations:
A = (T-d.b) / (c + d.a) D = b + a (T-d.b) / (c + d.a) CDT = A + D = b + (a + 1) (T-d.b) / (c + d. a) (wherein T represents the determined total transferrin content in the determined fraction (or in the separated fraction following separation);
A represents the actual asialotransferrin content in the sample;
D represents the actual disialotransferrin content in the sample;
CDT represents the actual total content of asialo-, monosialo- and disialotransferrins in the sample;
a and b are constants defining the correlation between A
and D in any serum sample; and c and d are constants specific to the determination step or separation method).
- g _ In a further aspect the invention provides a method for determining the content of a transferrin variant or combination of transferrin variants, preferably a CDT
variant or combination of CDT variants, in a body fluid for use in the assessment of alcohol consumption, said method comprising:
(a) subjecting a sample of said body fluid to a separation method capable of separating a fraction substantially free from tri- and higher sialylated transferrins;
(b) determining the total transferrin variant content in said fraction; and (c) determining the content of any transferrin variant or combination of transferrin variants, preferably the content of any CDT variant or combination of CDT variants, in said sample using an algorithm obtained in accordance with a method as herein described.
However, the separation step need not be provided if an alternative way to determine the target variants (asialo-, monosialo- and disialo- transferrins) is available. Furthermore, it is also possible to determine the content of only a portion of the target variants, so long as the portion of target variants is constant and reproducible.
Thus viewed from a broader aspect, the invention provides a method for determining the content of a transferrin variant or combination of transferrin variants, preferably a CDT variant or combination of CDT
variants, in a body fluid for use in the assessment of alcohol consumption, said method comprising:
(a) determining the content in a sample of said body WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 g -fluid of a transferrin variant or combination of transferrin variants in a fraction substantially free from tri- and higher sialylated transferrins;
(b) determining the total transferrin variant content in said fraction; and (c) determining the content of any transferrin variant or combination of transferrin variants, preferably the content of any CDT variant or combination of CDT variaf-~ts, in said sample using an algorithm obtained in accordance with a method as herein described.
In the assay in accordance with the invention, the measured transferrin variant content can be used to determine the actual levels of any transferrin variant or variants in the sample on the basis of a calibration derived from the correlation discovered for the levels of asialo- and disialotransferrins.
As will be generally understood, the determination step or separation method used in performing the assay in accordance with the invention will be the same as that used to produce the algorithm or calibration curve, such determination steps or separation methods being performed under the same set of conditions.
As used herein, the terms "determining" or "assessing" include both quantitation in the sense of obtaining an absolute value for the amount or concentration of transferrin variants) in the sample, and also semi-quantitative and qualitative assessments or determinations. An index, ratio, percentage or similar indication of the level or amount of transferrin variant(s), for example relative to total transferrin (i.e. all transferrin variants) may be obtained.
The assay method of the invention provides a convenient method for the determination of alcohol consumption by assaying the level of a transferrin variant or combination of transferrin variants in a body fluid, preferably a blood-derived body fluid, and may particularly find utility in the diagnosis and monitoring of alcoholism or alcohol abuse. Both asialo-and disialotransferrins, as well as a combination of asialo-, monosialo- and disialotransferrins have previously been shown to be a good indicator or marker for alcoholism or alcohol abuse, and by assaying the content of any of these in samples of body fluid, a distinction may be found between alcoholics and alcohol abusers and noh-alcohol abusers or social drinkers.
The body fluid used in the assay method of the invention may be any transferrin-containing body fluid for example, synovial fluid, amniotic fluid or cerebrospinal fluid, but will generally be blood or a blood derived sample. When this is the case, the sample used for analysis will preferably be cell-free and hence, either serum or plasma may be used. The sample may be treated prior to being used in the assay method of the invention, for example, it may be diluted by adding a buffer or other aqueous medium.
The purpose of the separation step used in the methods herein described is essentially to remove all.or substantially all tri-, tetra-, penta- and hexa-,. 25 sialotransferrins (non-target variants) from the sample, allowing the remaining target variants (asialo-, monosialo- and disialotransferrins) to be determined.
Where a determination step is referred to in relation to the methods herein described, this may in a preferred embodiment be a separation step. However, it may also be desirable in some circumstances to directly determine the content of the target variants) in a fraction of the sample without physically separating the fractions out.
For example, various immunoassay techniques are available which could allow detection or quantitation of target transferrin variants) in a body fluid. The antibody described in EP 0 605 627 is specific for a transferrin homolog found in alcoholics, or CDT, and therefore such an antibody may be used to determine the content of a target transferrin variant, or combination of transferrin variants. Provided that the antibody binds a constant fraction of the sample which is substantially free from tri- and higher sialylated transferrin it is possible to produce an algorithm in accordance with the invention.
Thus, in a particularly preferred aspect of the invention, the determination step is effectively a direct determination or direct measurement of the content of the target transferrin variants, or a combination of said target variants.
Transferrin preparations which may be regarded as substantially free from tri-, tetra-, penta- and hexa-sialotransferrins are those comprising less than 200, more preferably less than 10%, e.g. less than 5%
transferrin variants having three or more sialic acid residues per molecule.
Provided that the determination step or separation method is reproducible, separation of all asialo-, monosialo- and disialotransferrins or determination of the content of each is not required when carrying out the invention. Although possible to isolate all CDT
variants, clinically valuable results can be obtained by separating or determining only a fraction of these variants. What matters is that the separated or determined fraction of each variant is reproducible (i.e. essentially constant) for a given determination step under given conditions, or for a given separation method and given separation conditions. Quantitation (in the sense of obtaining an absolute value) of any or all of the CDT variants is not essential in carrying out the methods herein described.
Following separation (where separation is carried out), or in the case of a determination step, the fraction containing the desired target variants will typically comprise at least 60%, preferably at least 70 to 80%, e.g. 90 to 95% of the asialo- and monosialo-transferrin variants present in the sample prior to separation or determination. The disialotransferrin content of the separated or determined fraction will generally be at least 20%, preferably up to 60 to 700, particularly preferably 20 to 500, e.g. about 300 of the total disialotransferrin content of the sample prior to separation or determination.
In the separated or determined fraction containing the desired target variants, at least 40%, preferably at least 60%, e.g'. at least 70 to 80% of the transferrin molecules will carry a carbohydrate chain or a residue thereof. The separated or determined fraction will typically comprise less than 200, e.g. 10 to 150 asialotransferrin (or CFT), less than 5%
monosialotransferrin and 70 to 80%, e.g. about 750 disialotransferrin. An amount of trisialotransferrin, typically up to 20%, preferably up to 100, e.g. up to 50, can be tolerated without affecting the clinical value of the assay.
Thus for example the fraction to be determined or separated could consist essentially of asialotransferrin or it could could consist essentially of disialotransferrin, but in either case some or all of monosialotransferrin (and/or trisialotransferrin) could be present in the same fraction. Because the amount of monosialo (or trisialo-) transferrin is low relative to the amounts of asialo- and disialo- transferrin, this will not interfere significantly with the results of an assay in accordance with the invention.
Conveniently, ion exchange chromatography may be used to remove all or substantially all of the tri- and higher sialylated transferrins in any of the methods herein described. Ion exchange as a means of separating the various isotransferrin components is well known, and is described for example in US-A-4626355, Heil et al.
(supra) and WO 96/26444. Advantageously, an anion exchange chromatography step may be used, with the chromatography conditions (e. g. pH and ion binding strength) selected to permit retention of the desired transferrin variants (e.g. hexa-, penta-, tetra- and tri-sialo transferrin, and optionally some or all of the disialo fraction) .
Appropriate conditions e.g. buffering-capacity of the resin, sample/equilibration/elution buffer pH and/or ionic strength can readily be determined according to techniques known in the art, and according to the separation desired to be achieved. As is known in the art, prior to ion exchange, the sample may be treated with iron-containing buffer to saturate the iron-binding sites in the transferrin molecules in the sample.
Conveniently, according to techniques known in the art, chloride may be used as the counterion in the ion exchange procedure in order to achieve the desired separation. Thus, appropriate amounts of chloride ion present in the chromatography procedure necessary to achieve retention of the desired transferrin variants may be determined by routine experiments, and may depend on the precise conditions, batch of chromatography medium etc. The procedure can be monitored by isoelectric focusing or HPLC analysis, again according to standard techniques known in the art.
The ion exchange chromatography step may be carried out in any convenient manner known in the art according to choice, e.g. in a batch or column format. Likewise, the conditions may be selected to achieve the separation (i.e. depletion or removal) in any desired manner, for example by retaining the isotransferrin variants it is desired to remove (i.e. target variants), or by pre-treating the sample by ion exchange such that the "undesired" (i.e. non-target) variants do not absorb to the medium, and the remainder of the sample is separated and then eluted from the ion exchange medium.
Advantageously, the chromatography conditions are set to permit retention of the "undesired" transferrin variants.
As exemplary of ion exchange conditions which may be used, mention may be made of Whatman QASL anion exchange resin buffered at pH 6.3, which may be used to bind the trisialo and higher sialylated transferrins.
Alternatively, separation of target and non-target variants may be achieved using a binding ligand capable of binding selectively either to the target or non-target transferrin variants. Any binding ligand which has an affinity for the target or non-target transferrin variants may thus be used to separate the target variants from bther transferrin variants in the sample.
Preferably, the binding ligand may be a carbohydrate-binding ligand such as a lectin or mixture of lectins, e.g. as described in WO 99/00672. As noted in this earlier patent, 100% separation of CFT cannot always be achieved in practice. It has now been found that transferrin variants having a low carbohydrate content, in particular monosialo- and disialo-transferrins, bind with a low affinity to carbohydrate-binding ligands, such as lectins. In particular, it has been found that such variants bind to carbohydrate-binding ligands with a lower affinity than the transferrin variants having a higher or high carbohydrate content (i.e. the higher sialylated transferrins, e.g. tri-, tetra- and pentasialotransferrins). Separation methods using carbohydrate-binding ligands, for example lectins, are thus effective to remove higher sialylated transferrins (e. g. tri-, tetra-, penta- and hexa-sialotransferrins), but are generally less effective in removing lower sialylated transferrins (e.g. mono- and di-sialotransferrins). The result is an incomplete separation of CFT in which the separated "non-binding"
fraction will typically contain some or all of the mono-and disialotransferrins in addition to asialotransferrin (CFT). As noted previously, the assay method in accordance with the invention can tolerate incomplete separation of transferrin variants without compromising the clinical value of the assay.
WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 When the body fluid comprising transferrin variants is contacted with the carbohydrate-binding ligands, substantially all of the higher sialylated variants (tri-, tetra-, penta- and hexa-sialotransferrins) with carbohydrate side chains or remnants thereof are retained by the carbohydrate-binding ligands. The unbound fraction containing CFT, mono- and disialotransferrins may then be separated from the other variants and collected by any suitable means.
In a particularly preferred embodiment of the invention the separation method comprises the steps of contacting a sample of body fluid with a carbohydrate-binding ligand, e.g. a lectin or mixture of lectins, followed by separation of a fraction not binding to said ligand. The total amount of transferrin in the non-binding fraction may be determined directly by measuring the transferrin not bound by the carbohydrate binding ligand. Alternatively it may be determined indirectly by determining the amount of transferrin bound to the carbohydrate binding ligand and subtracting this from the total amount of transferrin present in the sample.
Generally, the direct approach is preferred.
Any carbohydrate-binding ligand or any combination thereof may be used to separate the target variants from other transferrin variants. This includes any ligand capable of binding to any carbohydrate or oligosaccharide or sugar structures. One or more carbohydrate-binding ligands may be used in the method of the invention. Where more than one ligand is being used, these may be used together or they may be used individually, for example, sequentially. The functional requirement of the carbohydrate binding ligand(s), rendering them suitable for use in the assay method of the present invention, is that they be capable of separating CDT from other transferrin variants.
Generally, the carbohydrate-binding ligand will be a protein, and very many such carbohydrate-binding proteins are known in the art and are widely described in the literature. The carbohydrate-binding protein may, for example, be an antibody, either polyclonal or monoclonal, or may be an antibody fragment for example F (ab) , F (ab' ) z or F (v) fragments . The antibodies or antibody fragments may be monovalent or divalent and they may be produced by hybridoma technology or be of synthetic origin, via recombinant DNA technology or chemical synthesis. Single chain antibodies could for example be used. The antibody may be directed or raised against any of the carbohydrate components or structures making up the Carbohydrate chains of glycosylated transferrin variants. Thus, for example, an antibody reactive with or selective for sialic acid residues might be used. Such an antibody is used in the Sialic Acid Deficient Enzyme Immunoassay (SDT-EIA) available from Medichem, Stuttgart, Germany and described in WO
97/19355.
More preferably, the carbohydrate-binding protein may be a lectin, used singularly or in combination with other lectins or with other types of carbohydrate-binding proteins, for example, antibodies. Any lectin known in the art may be used in the assay method of the invention and it may be of plant, animal, microbiological or any other origin. The literature is replete with references to different lectins which might be used, and many may be obtained commercially, for example, from Sigma.
Thus, included within the general term "lectin" as used herein, in addition to the classical plant lectins such as Concanavalin A (Con A), are carbohydrate binding proteins from microorganisms (for example, viral haemagglutinins) and higher organisms, including for example, invertebrates and mammals. Such mammalian carbohydrate binding proteins include selecting and other mammalian lectins or cell adhesion molecules (see for example Varki (1992) Current Opinion in Cell Biology 4:257-266).
Examples of suitable lectins are RCA-I (Ricinus communis agglutinin) which binds terminal galactose (Kornfeld et al. (1981) J. Biol. Chem. 256:6633) or Con-A (Concanavalin A), which is known to bind asparagine-linked oligosaccharides high in mannose. Other possibilities are Crotalaria juncea lectin which binds galactose residues (Ersson (1977) Biochim. Biophys. Acta 494:51-60), Wheatgerm agglutinin or Limulus polyphenus lectin which bind sialic acid (Mandal and Mandal (1990) Experientia 46:433-441) or Sambucus nigra agglutinin L
which binds NeuSAc/(«2-6)Gal/GalNAc (Shibuya et al.
(1987) J. Biol,'. Chem. 262:1596). As an example of a lectin derived from a micro-organism, a sialic acid specific lectin has recently been purified from the gut dwelling organism Helicobacter pylori (Lelwala-Guruge et al. (1993) APMIS 101:695-702).
Lectins of varying selectivity and specificity are known. Whereas some lectins may bind to a single sugar residue in a particular location on an oligosaccharide chain, for example RCA-I (from Ricinus communis) binds only to terminal galactose residues, some may bind to complex oligosaccharide determinants for example Sambucus nigra L which binds NeuSAc/(«2-6)Gal/GalNAc.
All are within the scope of the present invention.
Sialic acid binding lectins and other proteins represent a class of carbohydrate binding proteins of particular utility in the present invention (see the following, for example, for lists of suitable lectins and their sources: Mandal and Mandal(1990) Experientia 46:433-441); Zeng (1992) Z. Naturforsch, 47c:641-653 and Reuter and Schauer in Methods in Enzymology, Vol. 230, Chapter 10 at pages 196-198).
Particular mention may be made in this regard of Sambucus nigra L. Lectin, Sambucus sielbodiana lectin wheatgerm agglutinin, Maackia amurensis lectin, and E.
coli K99 lectin. S. nigra L. lectin is particularly effective when used on its own, although it may equally effectively be used in combination with other lectins eg. ConA.
WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 Some particular combinations of carbohydrate binding ligands useful for performance of the present invention are lectins from Helicobacter pylori and Ricinus communis; lectins from Ricinus communis and Sambuccus nigra; lectins from Crotalaria junctae and Sambuccus nigra; lectins from Crotalaria junctae and Helicobacter pylori and lectins from Ricinus communis and anti-sialic acid antibodies. The most preferred of the combinations are those which incorporate galactose-binding and sialic acid-binding ligands.
Followin~'the binding steps) a fraction may conveniently be collected which does not bind and which contains the CDT. Collection may be by any suitable means, for example, precipitation, centrifugation, filtration, chromatographic methods etc. Where different carbohydrate-binding ligands are used individually, different separation/collection formats may be used for each individual binding step.
Precipitation of carbohydrate-containing moieties in the sample may be achieved using lectins having known "precipitation" properties i.e. lectins capable of inducing precipitation of the moieties to which they bind. Combinations of lectins may advantageously be used for such a precipitation procedure, since differing lectin specificities increase the number of available binding sites. The non-binding (CDT) fraction may then readily be collected, for example by centrifugation or filtration to separate the precipitate.
In alternative embodiments, the carbohydrate binding ligand(s) may conveniently be immobilised to facilitate the separation and collection of the non-binding fraction. It is well known in the art to immobilise carbohydrate-binding ligands such as lectins for separation purposes, for example, in chromatographic columns, and any lectin affinity chromatography method known in the art could for example be used (see for example, Cummings (1994) Methods in Enzymology 230:66-86) .
WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 The carbohydrate-binding ligands may be immobilised by binding or coupling to any of the well known solid supports or matrices which are currently widely used or proposed for immobilisation or separation etc. These may take the form of particles, sheets, gels, filters, membranes, fibres or capillaries or microtitre strips, tubes or plates or wells etc and conveniently may be made of glass, silica, latex or a polymeric material.
Techniques for binding the ligand to the solid support are also extremely well known and widely described in the literature'. For example, the carbohydrate-binding ligands used may conveniently be coupled covalently to CNBr-activated Sepharose or N-hydroxysuccinimide-activated supports, optionally in the presence of low molecular weight haptens to protect the carbohydrate binding sites on the ligand. Other coupling methods for proteins are also well known in the art.
Batch separations using immobilised carbohydrate-binding ligands may be performed using a range of different formats which are known in the art.
In a different embodiment, although this is less preferred, the immobilised carbohydrate-binding ligands may be packed or arranged into a column. The body fluid comprising transferrin may be applied to the column and the transferrin variants therein contacted with the carbohydrate-binding ligands. The unbound fraction comprising CDT is separated from the bound fraction and collected.
The shape and geometry of such a column may vary depending upon the carbohydrate-binding ligands used.
For example, if lectins are used as the carbohydrate-binding ligands, at low lectin concentrations a long, thin column of immobilized lectins is preferred. At high lectin concentrations, column geometry is less crucial.
Columns may be constructed using any method known in the art. If lectins are to be used as the carbohydrate-binding ligands, the columns may be constructed in either glass tubes or preferably in disposable plastic pipettes of any desired capacity.
Smaller volumes may however be preferred due to economic considerations. Columns are preferably stored at around 4°C prior to use.
The column may be flushed through with an eluant to allow or facilitate collection of the unbound fraction, in which case the eluant should preferably be administered using a calibrated micropipette to ensure the correct volume is administered. The volume administered i~ preferably within 30 of the desired (i.e. calibration) volume, more preferably, within 1 or 2%. Since the rate of binding to oligosaccharides is comparatively slow, especially with plant lectins, it is preferable that slow flow rates are employed to maximise lectin/carbohydrate interactions. The eluant will generally be at a temperature within 5°C of the desired (calibration) value, e.g. 25°C, and more preferably within 1°C.
When using combinations of carbohydrate-binding ligands in a column format, either sequential columns using different ligands may be used or different ligands may be used in the same column material, either as a mixture or in a column comprising different layers, each layer having a different ligand.
In an alternative embodiment, the carbohydrate-binding ligand may be immobilised on a particulate solid phase, for example, latex, silica or polymer beads. To aid manipulation and separation, magnetic beads may be used. The term "magnetic" as used herein means that the support is capable of having a magnetic moment imparted to it when placed in a magnetic field. In other words, a support comprising magnetic particles may readily be removed by magnetic aggregation, which provides a quick, simple and efficient way of separating the fractions following the carbohydrate binding step.
Thus, the magnetic particles with non-target variant moieties attached may be removed onto a suitable surface by application of a magnetic field, for example, using a permanent magnet. It is usually sufficient to apply a magnet to the side of the vessel containing the sample mixture to aggregate the particles to the wall of the vessel and to collect the remainder of the sample, which will comprise the "non-binding, CDT-containing fraction" which may be returned for subsequent analysis.
Especially preferred are superparamagnetic particles, which include for example those described by Sintef in EP-A-106873, as magnetic aggregation and clumping of the particles during the reaction can be avoided. Magnetic particles are commercially available from a number of sources, including for example, Advanced Magnetics Inc., (USA), Amersham (UK), Bang Particles (USA), and Dynal AS (Oslo, Norway).
Functionalised coated particles for use in the present invention may be prepared by modification of the beads, for example according to US patents 4,336,173, 4,459,378 and 4,654,267. Thus, beads, or other supports, may be prepared having different types of functionalised surface, for attachment of a desired carbohydrate-binding ligand.
Separations based on centrifugation and/or filtration are convenient. In a preferred embodiment a centrifuge tube (e. g. Eppendorf tube) and "filter cup"
format may be used, and such formats are readily commercially available, for example from Millepore.
Thus the sample and carbohydrate-binding ligand may be added to the cup in the tube and allowed to bind. The tube (and cup) is then spun, and the non-binding supernatant collects in the tube. The carbohydrate-binding ligand may be such as to induce precipitation of the bound carbohydrate moieties or it may be immobilised, for example as a slurry e.g. a gel or on particles. In either case, the bound carbohydrate binding fraction is retained in the cup.
As a variation of such a "tube and cup"
arrangement, the cup may be provided with one or more WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 "discs" or filters which carry immobilised carbohydrate-binding ligands.
Following the determination step or the separation step, the total content of transferrin in the separated fraction is determined. This may be done by any standard procedure known in the art for assay of transferrin, for example, by any standard immunoassay technique, e.g. an ELISA or radio-immunoassay technique.
Methods for determining transferrins are described for example in US-A-4,626,355 (Joustra).
An example of an ELISA method could include a sandwich assay in which an immobilised antibody specific for transferrin variants which are substantially free from tri- and higher sialylated transferrins is contacted with the sample, and then enzyme-labelled anti-transferrin antibodies (available from Dako AS, Denmark) are used to detect the bound transferrin. A
preferred such immobilised antibody is the antibody described in EP 0 605 627 and in such a case it will be appreciated that the separated fraction will be the fraction of antibody-bound transferrin and that the enzyme signal will be proportional to the total transferrin content of that fraction, allowing the total transferrin to be determined in that fraction.
Many commercial assays for transferrin are available and have been described in the literature.
For example an RID (radio immuno diffusion) assay based on the method of Mancini is available from Hoechst (see Mancini et al. Immunochemistry, 2:235-254 (1965)). A
rocket immuno electrophoresis method is described by Laurell in Scand. J. Clin. Lab. Invest. 29 (Suppl. 124):
21-37 (1972). Particular mention may also be made of the particle-based immunoassay method of Miiller et al.
in Lab. Med. 1:278 (1991). This is a very sensitive technique, based on an enhanced turbidometric method which uses a turbidometric signal but is more sensitive than traditional turbidometric methods.
For either turbidimetric or nephelometric transferrin determination, opacity will generally be generated by contacting the separated fraction or an aliquot thereof with an anti-transferrin antibody or antibody fragment, e.g. a rabbit anti-human transferrin antibody such as is commercially available from Dako of Copenhagen, Denmark. The Dako antibodies are specific to transferrin and show no cross reactions with other blood proteins that may be present in the eluate. The quantity of antibody used should of course be optimised against transferrin containing standard samples as opacification irises from the hook effect whereby multiple transferrin binding generates the opacification centres.
In the case of the "tube and cup" embodiment described above for example, the anti-transferrin antibodies may simply be added to the tube after centrifugation.
As in routine turbidimetric and nephelometric assays, a polymeric opacification enhancer, such as polyethyleneglycol, is preferably also added to the eluate.
In determining transferrin content using such measuring techniques, a kinetic reading mode may of course be used.
Before the nephelometric or turbidimetric determination is made, the fraction, antibody and enhancer may be incubated for a short period, e.g. 5 minutes to an hour for end-point measurements, preferably about 10 minutes.
The light used in the determination of opacification should have an appropriate wavelength. In this regard we have found that use of a 405 nm filter, or more preferably a 340 nm filter, yields particularly good results.
Where a separation method is to be avoided and a direct determination step is instead to be used, a suitable method is to take advantage of a proximity interaction technique. Most preferably, in this embodiment of the invention, a specific binding partner for the target transferrin variants is utilised in a proximity assay to obtain a direct determination of the amount of the target variants (asialo-, monosialo- and disialotransferrins) in the fractions) to be determined. An example of a suitable specific binding partner is the anti-transferrin antibody which reacts selectively with transferrin homologs found in alcoholics but not in non-alcoholics, as disclosed in EP 0 605 627.
Another means of producing a suitable specific binding partner with appropriate specificity for the target transferrin variants to be determined is to raise an antibody against a peptide immunogen which mimicks the N-glycan binding site on the transferrin molecule, for example if the peptide sequence corresponds to the amino acid sequence of human transferrin. The amino acid sequence of human transferrin is known and is published for example in Yang et al., Proc. Natl. Acad.
Sci. 81: 2752-2756 (1984) or accession number PO 2787 Swiss Prot database.
Examples of suitable techniques which allow for detection of a signal due to proximity interaction between molecules are described in the literature. Such techniques include fluorescence polarisation immunoassay technology (FPIA), fluorescence quenching techniques, proximity scintillation assays and EMIT technology.
FPIA techniques are described for example in Dandliker et al., Immunochemistry 7: 799-828, (1970), and in Wei et al., Anal. Chem. 65: 3372-3377 (1993)).
Fluorescence quenching techniques are described for example in US-A-3,996,345 of Ullmann et al., in which one binding partner carries a fluorescent residue and the other carries a quencher.
Proximity scintillation assays are described for example in US-A-4,568,649 and EP 0 154 734 of Bertoglio-Matte. In these assays, one of the binding partners emits a short-range energy-rich radioactive signal, WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 typically emitting (3-rays, and when proximity interaction with the other binding partner occurs, a fluorophore linked to the second binding partner is excited by the radioactive energy and a fluorescent signal is generated.
A further, more complex type of fluorescent proximity assay was described in US-A-5,763,189 of Buechler et al. which is based upon the measurement of Stokes shift (difference in wavelength of light which is emitted as compared to the excitation wavelength).
The EMIT technology is described for example in US-A-3,852,157 of Rubenstein et al. and this technique relies on competition between analyte molecules and enzyme-labelled analyte analogs (the "reactant") for a receptor (the "specific binding partner") in the assay solution.
Further signal detection techniques include turbidimetry and nephelometry as described hereinbefore in relation to separation methods.
In general, besides the sample under evaluation, calibration samples with known transferrin contents will also be assessed in the performance of the assay method of the invention. Such determinations can be used to plot a calibration curve from which the transferrin content of the sample under evaluation may be determined. Preferably calibration samples having transferrin contents of up to 0.05 mg/ml (e. g. 0.002, 0.01, 0.02 and 0.03 mg/ml) will be used.
In the assay method of the invention the total transferrin content of the determined or separated fraction containing the target variants will preferably be determined. Using an algorithm as herein described this can then be used to determine with a high degree of accuracy the content of asialo- or disialo-transferrin, or CDT content of the sample. The content of asialo- or disialo-transferrin or CDT may be determined as a percentage of total transferrin. This may be a more precise marker for alcohol consumption than total transferrin, and a threshold value, for example lo, may be set. Alternatively, the presence of any transferrin variant may be assessed as an actual concentration (i.e.
a mass per unit volume).
Viewed from a further aspect, the invention provides a kit for a diagnostic assay according to the invention, said kit comprising:
means for subjecting a sample of body fluid to a determination step or separation method capable of producing or determining the content of a fraction substantially ;free from tri- and higher sialylated transferrins;
means for the detection of transferrin; and means for determining the content of any transferrin variant or combination of transferrin variants in a sample of body fluid subjected to said separation method or determination step.
Conveniently, the kit may also comprise a transferrin standard or standards for reference. Thus, in one preferred embodiment, the kit of the invention may comprise:
at least two transferrin solutions having known asialo- and disialo- concentrations;
means for subjecting a sample of body fluid to a separation method or determination step capable of producing or determining the content of a fraction substantially free from tri- and higher sialylated transferrins;
means for the detection of transferrin; and means for determining the content of any transferrin variant or combination of transferrin variants in a sample of body fluid subjected to said separation method or said determination step.
The invention will now be illustrated by the following non-limiting Examples and the accompanying figures in which:
Figure 1 shows the high correlation (R2 - 0.9153) between the concentration of asialo- and disialotransferrin variants in a serum sample determined in accordance with the method of Example 1.
Figure 2 shows the high correlation (R2 - 0.9336) between the o of asialo- and disialotransferrin variants in a serum sample determined in accordance with the method of Example 1.
Figure 3 shows separation of transferrin isoforms by HPLC. ' Figures 4A and 4B show correlation of asialo vs monosialo, in A determined in o transferrin, in B
determined in ~.g/mL.
Figures 5A and 5B show correlation of asialo vs disialo, in A determined in o transferrin, in B
determined in ~.g/mL.
Figure 6 shows the correlation of (asialo-, monosialo- and disialo- transferrin) with asialo transferrin, determined in mass units (~,g/mL) .
Figure 7 shows the correlation of (asialo-, monosialo- and disialo- transferrin) with disialotransferrin, determined in mass units (~g/mL).
Figure 8 shows the correlation of asialo transferrin with (monosialo- and disialo-) transferrin, determined in o transferrin.
Figure 9 shows the correlation of asialo- with trisialo- transferrin, determined in o transferrin.
Figure 10 shows the correlation of (asialo-, monosialo- and disialo-transferrin) with asialotransferrin, determined in o transferrin.
Figure 11 shows the correlation of (asialo-, monosialo- and disialo- transferrin) with disialotransferrin; determined in o transferrin.
Example 1 Reference method - Determination of asialo-, monosialo-, disialo- and trisialo-transferrin content of serum samples The transferrin variant content of lipid stripped, iron treated serum samples was analysed using a combined HPLC-RIA method. The levels of the different sialic acid isoforms were determined first by separation on HPLC with an ibn exchange column. The asialo-, monosialo-, disialo- and trisialotransferrin fractions were collected in separate tubes. The higher isoforms, tetrasialo-, pentasialo- and hexasialo-transferrins, were collected in a separate tube. The a of the different fractions was first determined using HPLC, then the concentration of the fractions was determined on a gamma counter using an immunoassay procedure as outlined below.
Reagents Dextransulfate sodium salt Calcium chloride dihydrate Ferric chloride, FeC13.6H20, min. 99.0%
Acetic acid > 990 2-propanol Nitrilotriacetic acid trisodium salt monohydrate >980 BisTris (bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane) > 99.50 NaCl p.a.
2 M NaCl, p.a.
2 M NaOH, p.a.
5o Acetic acid 75o Acetic acid Mobile phase A: 20mM BisTris buffer, pH 6.5 Mobile phase B: 20mM BisTris buffer, pH 6.5, 0.5M NaCl Mobile phase C: 20mM BisTris buffer, pH 5.8 Mobile phase D: water Equipment - 2 ml syringe - 0.2 ~m filter for filtering serum samples (Gelman Acrodisc 13) - Column: Pharmacia Resource Q 1 mL, Code 17-1177-O1 or Pharmacia Source 15Q 1 mL
- Pre-column: HP Pre-column cartridge holder 4.Omm inner diameter, filled with HQ50 in slurry form - Transferrin, in solution and labelled with lzsI
- Anti-transferrin antibody (raised in rabbit) solution - Sheep anti-rabbit antibody (decanting solution) - Calibrators 0, 5, 20, 50, 100 and 300 U CDT/L
(1 U/L = 0.034 ~.g/ml) - Transferrin calibrators (based on Seronorm calibrated serum supplied by Sero AS, Norway, diluted in mobile phase A) 0, 0.05, 0.10, 0.15, 0.25, 0.50, 0.75, 1.0, 1.5, 2.5 ~,g/ml - HPLC Degaser: Degaser Mod. G1322A
- HPLC Pump: Quaternary pump Mod. G1311A
- HPLC Injector: Autosampler Mod. G1313A, modified with a 900,1 injector loop - HPLC Thermostat: Column thermostat Mod. G1316A, with a Reodyne multi-port - HPLC Detector: Diode Array Detector Mod. G1315A
Data Handling: HP ChemStation -- pH meter: Hanna Instruments 8417 - Centrifuge: Minifuge RF, Z-924 - Gamma counter: RIASTAR
- Fraction collector: GradiFRAC from Pharmacia HPLC
150.1 of each serum sample was added to 30.1 FeNTA (an aqueous solution containing 2.751 g/1 nitrilotriacetic trisodiummonohydrate and 2.703 g/1 FeC13.6H20 adjusted to a pH of 6.5 using 2M NaOH) and vortexed. The resulting solution was then added to 101 dextran sulfate (20 mg/ml) and 10.1 potassium chloride (147 mg/ml). The solution was cooled to 2-8°C for 30 mins. The sample was then centrifuged at 3800 rpm for 10 mins. 150,1 of the supernatant was pipetted out and added to 900.1 HPLC
buffer A. The sample was filtered on an acrodisc filter and then injected into the HPLC in an aliquot of 8001.
The transferriri isoforms were separated by an ion-chromatographic gradient method. The transferrin was selectively detected spectrophotometrically at 470 nm by the HP ChemStation.
Five fractions of the transferrin were collected from the HPLC into tubes in exact amounts. The fractions asialo- to trisialotransferrin were collected separately. The tetrasialo- to hexasialotransferrin were collected as a separate fraction. The asialo- and monosialo- fractions were not further diluted. The disialo- and trisialo- fractions were diluted 1:1 and the fraction containing the tetrasialo- to hexasialotransferrins was diluted 1:4 with mobile phase A.
To quantify the transferrin content of each isolated eluted fraction, the steps D1-D8 in the following immunoassay procedure were performed. In relation to the asialo fraction, in D8 the concentration was determined using calibrators from 0-2.5~.g/ml. In relation to the remaining fractions the steps D1-D8 were performed with the CDTect-calibrators.
Immunoassay procedure D1: 5001 calibrator is pipetted out. The calibrators are dilutions of human transferrin (obtained from WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 Intergen) in PBS containing 1% BSA, O.lo Tween 20 at pH 7.0) D2: 5001 sample is pipetted out D3 : 50.1 transferrin lzsl is added to the sample . The lzsl transferrin solution is made by dilution of lzsl labelled human transferrin (obtained from Isopharma AS, Kjeller, Norway) to a concentration suitable for gamma-counting in an aqueous buffered solution containing 0.037 M disodiumhydrogen phosphatedihydrate, 0.013 M sodiumdihydrogen phosphatemonohydrate, to BSA, 0.1% Tween 20 and 0.03% Patent Blue.
D4: 501 antibody is added. The antibody solution is made from rabbit anti human transferrin antiserum (obtained from BioCell, product No. 01090) diluted 1:450 in 0.4 M sodium phosphate buffer with to BSA, O.lo Tween 20 at pH 7Ø
D5: 2000.1 decanting suspension is added. The decanting suspension is a solution of secondary anti rabbit antibodies (obtained from Pharmacies &
Upjohn, product No. 30-3794-00) D6: incubate for 1 hour D7: centrifuge 10 mins at 1500xg and remove supernatant D8: determine radioactivity The concentration and o distribution of transferrin variants determined in a given serum sample is shown in Table 1 below (1501 serum is diluted to 1.1 ml and 800,1 is injected on the HPLC; dilution factor:
1.1/0.15*1/0.8 = 9.17):
Table 1 asialo mono- di- tri- tetra higher fiPLC fraction volume4 2.5 5 5.5 27.5 (ml) Sample volume (~l) 500 500 250 250 125 Conc. in RIA sample 0.29 0.29 0.87 1.20 2.85 (/a.l/mg) Conc. corrected for 0.29 0.29 1.73 2.41 11.41 sample volume (~1/mg) Conc. per ml 1.14 0.73 8.67 13.24 313.66 fraction (~g/ml) Conc. in serum 10.47 6.66 79.50 121.45 2876.26 (~1/mg) % CDT-RIA 0.34 0.22 2.57 3.92 92.95 Example 2 Correlation between asialo- and disialotransferrins and lack of monosialotransferrin Using the HPLC/RIA method described in Example l, monsialotransferrin levels were found to be very low.
In addition, asialo- and disialotransferrin levels were found to correlate as follows:
D = A.a + b (wherein D represents disialotransferrin content of a serum sample;
A represents asialotransferrin content of a serum WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 sample;
a = 22.2; and b = 36 mg/1) This represents a squared correlation coefficient of 0.9153 (see attached Figure 1).
Using relative units (% total transferrin coefficients), the following values for a and b were determined:
a = 22.25 ' b = 1.05% (0.0105) This represents a squared correlation coefficient of 0.9336 (see attached Figure 2).
Example 3 Determination of algorithm to calculate levels of asialotransferrin, disialotransferrin and CDT in a serum sample At least two solutions having known levels of asialo-and disialotransferrins are subjected to a separation method capable of removing substantially all tri- and higher sialotransferrins (e.g. based on lectins or ion exchange matrices). Thereafter the total transferrin content of the isolated fraction of transferrin is determined (e.g. by a radioimmunoassay method for transferrin quantitation). Based on the previously determined correlation between the level of asialo- and disialotransferrins and the low level of monoasialotransferrin, the following relations can be established:
T1 = c.Al + d.Dl T2 - c.A2 + d.D2 WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 T3 - c.A3 + d.D3 etc.
(wherein T1, T2, T3, etc. represent the measured total transferrin content of each sample following separation*;
A1, A2, A3, etc. represent the known contents of asialotransferrin in each sample;
D1, D2, D3, et,c. represent the known contents of diasialotransferrin in each sample; and c and d are constants).
* these may be measured in mass units or relative to the total transferrin content of the sample.
Calculation of the content of asialotransferrin, diasialotransferrin and CDT in any sample from the partial measurement of T:
For any sample tested:
T = c.A + d.D
and D = a.A + b (wherein T represents measured total transferrin content;
A represents actual asialotransferrin content in the sample;
D represents actual disialotransferrin content in the sample; and a, b, c and d are each constants).
It follows that:
WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 A = (T-d.b) / (c + d. a) ;
D = b + a(T-d.b)/(c + d.a); and CDT = A + D = b + (a + 1) (T-d.b) / (c + d.a) (where CDT is the total content of asialo-, monosialo-and disialotransferrin in the sample).
Example 4 Quantitation of transferrin content by means of anion exchange and lectin binding 20 ~.1 of a serum sample is mixed with 50 ~.l of a solution of 10 mM bis-tris, 3.1 mM sodium azide, 0.05%
Tween 20, 1 M HCl ~d pH 7.0, 0.8 mM Tris base, 0.15 mM
FeCl3, 0.15 M sodium citrate and 0.4 mM malefic acid.
The resulting solution is added to 0.5 ml of 250 preswollen Whatman QA52 ion exchange resin suspended in 20 mM Bis-Tris buffer ((2-hydroxy)amino-tris(hydroxymethyl)methane) pH = 6.3. The chloride content of the medium is carefully adjusted to retain substantially all transferrin molecules with more than two sialic acid residues (this may be monitored by HPLC
or isoelectric focusing). Thereafter, 0.25 ml of a 25%
suspension elderberry bark lectin (Vector Laboratories, US) is added (this ensures a more complete mopping up of sialylated transferrin molecules), and the suspension is mixed gently. The suspension is thereafter filtered by centrifugation in a Millipore Ultra-Free UFC3 OHV filter cup, and the filtrate is collected. 200 ~.1 of the filtrate is mixed with 200 ~,l of a transferrin antibody (Dako) solution diluted 1:10 in 0.27 M Tris, 4.5% PEG
8000, 4.3 mM sodium azide pH = 7.4. The concentration of transferrin in the filtrate is determined by interpolating the nephelometric signal in a standard curve constructed from standards of known concentrations of human transferrin.
With this separation method, c = 0.93 and d = 0.45.
Example 5 Quantitation of transferrin content by means of anion exchange 20 ~.l of a serum sample is mixed with 50 ~.l of a solution of 10'mM bis-tris, 3.1 mM sodium azide, 0.050 Tween 20, 1 M HCl ad pH 7.0, 0.8 mM Tris base, 0.15 mM
FeCl3, 0.15 M sodium citrate and 0.4 mM malefic acid.
The resulting solution is added to 0.5 ml of 250 preswollen Whatman QA52 ion exchange resin suspended in mM Bis-Tris buffer ((2-hydroxy)amino-tris(hydroxymethyl)methane) pH = 6.3. The chloride content of the medium is carefully adjusted to retain substantially all transferrin molecules with more than 20 two sialic acid residues (this may be monitored by HPLC
or isoelectric focusing). The suspension is thereafter filtered by centrifugation in a Millipore Ultra-Free UFC3 OHV filter cup, and the filtrate is collected. 200 ~.1 of the filtrate is mixed with 200 ~.l of a transferrin antibody (Dako) solution diluted 1:10 in 0.27 M Tris, 4.5o PEG 8000, 4.3 mM sodium azide pH = 7.4. The concentration of transferrin in the filtrate is determined by interpolating the nephelometric signal in a standard curve constructed from standards of known concentrations of human transferrin.
Example 6 Quantitation of transferrin content by means of immobilised lectiwfrom Sambuccus nigra in a column format.
a. 10 ~,l serum samples are mixed with 0.5 ml binding buffer 20 mM Tris-HC1 buffer with pH = 7.5 containing 150 mM sodium chloride.
b. Each diluted serum sample is passed through a column of~0.5 ml agarose elderberry bark (Sambuccus nigra) le~ctin (supplied by Vector Laboratories, Burlingame, USA) suspended in 20 mM Tris-HCl buffer with pH = 7.5 containing 150 mM sodium chloride, and another 1.0 ml of the same buffer is passed through the column.
c. Mix 200 ~.1 of the eluted solution with 200 ~,1 of an anti-transferrin antibody solution comprising 0.27 M Tris, 4.5% PEG 8000, 4.3 mM sodium azide, 1:10 dilution of Dako anti-human-transferrin antibodies Q0327, and HC1 to pH = 7.4.
d. Read the turbidimetric/nephelometric signal.
With this separation method, c = 0.98 and d = 0.08.
Exam 1p a 7 Equipment - Columns (7mm internal diameter) containing 0.5m1 POROS HQ50 (Perseptive Biosystems) between polyethylene upper and lower porous frits (Porex, Atlanta, Ga, USA) - columns available from Pierce Company, USA
- Four calibrators**
- Pipettes covering volumes from 4~,1 to 3m1 Alternatively: multipipettes for volumes 1001, 200,1, 2m1 and 3m1 WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 - Racks (for tubes (75 x l2mm) - Microtiter plates -- Reader for microtiter plates, 405nm filter ** Prepared using human normal serum. See Table 2 for dilutions Table 2 Calibrators (mg/ml) Human normal serum Solution 2 containing 2.17 mg transferrin/ml C1:0.002 100.1 C3 900.01 C2:0.01 4.6.1 995.4,1 C3 :0.02 10.1.1 1089.9~C1 C4:0.03 13.81 986.2.1 (Calibrator C1 is produced by dilution of calibrator C3 with Solution 2).
Reagents - Solution 1 lOmM BisTris (bis(2-hydroxyethyl)amino-tris-(hydroxymethyl)methane) 3.lmM Sodium Azide 0.05% Tween 20 1M HC1 ad pH 7.0 0.8mM Tris base (Tris(hydroxymethyl)aminomethane) 0.15mM FeCl3 0.15mM Sodium Citrate 0.4mM Malefic acid deionized HZO a.s.
Solution 2 50mM BisTris (bis(2-hydroxyethyl)amino-tris-(hydroxymethyl)methane) WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 3.lmM Sodium Azide 0.05% Tween 20 1M HCl ad pH 6.00 approx 3 mM NaCl added deionized H20 ,~.s.
Turbidimetric reaaent 900.1 0.3M Tris/PEG pH 7.4 100.1 Rabbit anti-serum transferrin (Dako) The 0.3M Tris/PEG pH 7.4 comprises:
0.3m Tris. HC1 6% Polyethylene glycol (PEG 8000) 3.lmM Sodium Azide 2M NaOH ~d pH 7.4 deionized HZO a.s.
Preparation of columns:
Use one column for each sample to be tested. Elute surplus transport buffer by removing first the top and thereafter the bottom stopper, discard and eluate.
Prepared columns should be used within 2 hours.
Sample testina procedure - Sample procedure 1. Add 100,1 serum to 500.1 solution 1 in a test tube. Mix.
2. Incubate for 5-10 minutes at ambient temperature.
- Column separation 1. All solutions added must elute freely from the column.
2. Add 500.1 incubated sample to a column.
3. Let the sample sink into the top filter before adding l.Oml of solution 2.
4. Let solution 2 sink into the top filter.
Change tubes below columns. Solution eluted up to this point should be discarded. Add 2.0m1 of solution 2 to each column. Collect 2m1 eluate 2.
- Measurement 1. Add 200,1 of each calibrator, and 200,1 of eluate 2 from the column separation to separate wells of a microtiter plate. Read at 405nm.
2. Add 100.1 turbidimetric reagent to each well.
3. Incubate for 15 minutes at ambient temperature.
4. Read Y~esults using a reader with 405nm filter and subtract the background calculated in step 1.
5. Establish a calibration curve using non-linear regression.
6. transferrin content in the serum sample is calculated from the calibration curve.
With this separation method, c = 0.98 and d = 0.66.
Example 8 Determination of asialo- and disialo- transferrin, and a combination of asialo-, monosialo- and disialo-transferrin, using a combined HPLC-RIA method.
A further experiment was carried out to determine the correlation between 1) asialo- and monosialo- transferrin isoforms in serum samples, 2) asialb- and disialo- transferrin isoforms in serum samples, 3) (asialo-, monosialo- and disialo-) and asialo-transferrin isoforms in serum samples, and 4) (asialo-, monosialo- and disialo-) and disialo-transferrin isoforms in serum samples.
These experiments were carred out to test the reliability of the relationship between the content of asialo-, mono sialo- and disialo- transferrins (in the above combinations 1-4) in serum samples from a number of different individuals.
REAGENTS, MATERIALS, INSTRUMENTS AND EQUIPMENT
The experiment was carried out using the same reagents, materials, instruments and equipment as described in Example 1.
EXPERIMENTAL
Transferrin in lipid stripped, iron treated serum was analysed on two combined methods, HPLC and RIA. The different sialic acid isoforms were determined, first by separation on a HPLC with a ion exchange column. The o of the different fractions are first determined on the HPLC, then the concentration of the fractions are determined on a gamma counter by using parts of the CDTectTM
RESULTS AND DISCUSSION
Fourteen samples of serum were collected from 14 individuals and in each of these samples, the asialo-, mono- and disialo - transferrin fractions were separated and collected individually. For each sample, the concentration of each fraction was determined as percentage of total transferrin using the RIA method (%CDT-RIA) and HPLC method (%CDT-HPLC) as described in Example 1. The concentration of the asialo-, mono- and di-sialo transferrin was also determined in ~.g/mL for the RIA method'(CDT RIA ~.g/ML). These results are shown below in Table 3.
WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 CDT-RIA ~ CDT
CDT-HPLC RIA
~g/m sialomonodi tri etra+sialomonodi ri etrapentasialomonodi SB 0.18 0.3 1.695 92.820 0.151.285 75.4818.1 0.4 0.525.4 CDT 0.34 0.49.6 7.0487.520.180.15.82 5.3972.2419.22.68 0.779.2 4 0. 0.43. 8 88 0 0 1. 7 76. 14 . 0. .
18 08 .46 . .24 32 . 59 . 36 65 04 123 0.33 0.39.11 6.7788.410.190.14.86 6.9859.420.42 .84 0.9910.6 2 1.07 0.7 10.68.0779.590.930.389 8.8 58.921.98 .92 1.2418.9 122 0.43 0 . 6.4288 0. 0 3 5 67 3 .26 0. 13 . 61 . 35 . . .24 . . 64 .4 7 0. 0. 1 9. 87. 0. 0 1 8 68 1 .41 1 5 13 39 . 93 74 03 . . .41 .23 .21 .23 .
126 0.51 0.345.52.46 89.170.530.155.323.9263.326.76 .34 1.2219.7 127 0 0 1 . 93 0 0 1 .49 68 4 . 0 7 . . . 66 . . . . . . 83 . .
BL 0 0 1 5 92 0 0 0 . 76. 18 0 0. 3 .41 . . . . . 12 79 . 85 .
129 0.41 0.45.41 .37 90.370.350.19.59 3.9967.423.47 .23 1.3413.2 130 0.88 0.718.097.2 83.130.910.247.326.9470.0114.58.55 1.2514.2 136 0.13 0.191.3 1.3497.040 0.131.04.63 2.961.25 .44 0.664.6 125 0.35 0.49.87 7.1687.130.110.273.237.4868.80.12 .94 1.3 12.9 Figure 3 shows separation of transferrin isoforms by HPLC. The separation of monosialo and disialo is not complete, as can be seen between 9 and 10 min.
Figures 4A and 4B show correlation of asialo vs monosialo, in A determined in o transferrin, in B
determined in ~,g/mL. The squared correlation coefficient based on percentage determinations (Fig. 4A) was 0.5235. Based on mass units (Fig. 4A) the squared correlation coefficient was 0.3339. The graphs are produced using data from the RIA method.
Hence the asialo vs monosialo transferrin did.not show a good correlation for either of the determinations. One of the reasons for this might be that for the higher asialo values the disialo peak seems SUBSTITUTE SHEET (RULE 26) to overlap the monosialo. This can be seen in Figure 3.
Figures 5A and 5B show correlation of asialo vs disialo, in A determined in o transferrin, in B
determined in ~.g/mL. The graphs are produced using data from teh RIA method.
The squared correlation coefficient based on percentage determination (relative units) was 0.9501 (see Fig. 5a). Based on mass units (Fig. 5B) the squared correlation coefficient was 0.8603.
The correlation of asialo- vs disialo- transferrin was as expected better for the % determination than for ~g/mL. This is due to the correction for high and low total transferrin for high and low total transferrin content, in the % isoform determination.
Figure 6 shows the correlation of (asialo-, monosialo- and disialo- transferrin) with asialo transferrin, determined in mass units (~,g/mL). The squared correlation coefficient was 0.8797. The graph is produced using data from the RIA method.
Figure 7 shows the correlation of (asialo-, monosialo- and disialo- transferrin) with disialotransferrin, determined in mass units (~g/mL).
The squared correlation co-efficient was 0.9176. The graph is produced using data from the RIA method.
At present, the precision of asialotransferrin determination is lower than e.g. disialotransferrin (due to much lower concentration), and therefore the correlation coefficient for asialotransferrin is lower.
However the correlation coefficient for asialotransferrin is much higher than had previously been thought possible.
Example 9 Determination of asialo, monosialo, disialo and trisialo, and combinations of asialo, monosialo and disialo transferrin, using a combined HPLC-RIA method.
A further experiment was carried out to determine the correlation between 1) asialo- and (monosialo- and disialo-) transferrin isoforms in serum samples, 2) asial,b- and trisialo- transferrin isoforms in serum samples, 3) (asialo-, monosialo- and disialo-) and asialo-transferrin isoforms in serum samples, and 4) (asialo-, monosialo- and disialo-) and disialo-transferrin isoforms in serum samples.
These experiments were carred out to test the reliability of the relationship between the content of asialo-, mono sialo- and disialo- transferrins (in the above combinations 1-4) in serum samples from a number of different individuals.
REAGENTS, MATERIALS, INSTRUMENTS AND EQUIPMENT
The experiment was carried out using the same reagents, materials, instruments and equipment as described in Example 1.
EXPERIMENTAL
Transferrin in lipid stripped, iron treated serum was analysed on two combined methods, HPLC and RIA. The different sialic acid isoforms were determined, first by separation on a HPLC with a ion exchange column. The o of the different fractions are first determined on the HPLC, then the concentration of the fractions are determined on a gamma counter by using parts of the CDTectTM.
RESULTS AND DISCUSSION
Twenty six samples of serum were taken from 26 individuals. However the mono- and disialo-transferrin fractions were collected together as a single fraction.
The concentrations of each fraction were determined by the RIA and HPLC methods respectively and the results are shown in Table 4 below:
% CDT-RIA % CDT
HPLC
~g/mL
sialo+ tri etra entasialo + tri etra penta di di ampl a 1.5 2.1 5.5 63.2 17.81.3 11.74.6 3.4 19 ample 1.2 0.5 4.2 67.8 15.21.4 11.34.7 4.6 17.1 ample 0.5 3.7 6.3 73.5 16 0.6 3.8 6.1 2.8 16.7 ample 0 ~2 4.6 75.4 18 0.6 2.6 4.7 0.2 22 10 ample 0 2.6 3.1 70 4.4 0.4 3 3.1 4.7 8.8 ample 0.5 4.1 5.1 69.4 0.8 0.4 3.9 4.8 7.1 3.7 ample 0 1.6 6.12 76 16.30.5 1.3 5.7 7.6 14.9 ample 0 2.8 5.5 73.6 18.10.4 2.7 4.7 4.3 17.8 ample 0 2.9 5.4 73.3 18.40.2 3.1 4.9 2.4 19.4 1 ample 0.3 3.8 6 73.6 16.30.7 3.8 5.3 1.8 18.4 ample 0 1.5 2.2 74.1 2.2 0.5 1.5 2.2 5.6 0.3 ample 0.4 3.1 5.9 72 18.50.8 3.4 5.6 1.3 18.9 Figure 8 shows the correlation of asialo 20 transferrin with (monosialo- and disialo-) transferrin, determined in o transferrin. A reasonable correlation was found. The squared correlation coefficient was 0.8596. The graphs are produced using data from the RIA
method.
Figure 9 shows the correlation of asialo- with trisialo- transferrin, determined in % transferrin. As expected, no correlation was found. The squared correlation coefficient was very low, at 0.01754. The graph is produced using data from the RIA method.
Figure 10 shows the correlation of (asialo-, monosialo- and disialo-transferrin) with asialotransferrin, determined in o transferrin. The squared correlation coefficient was 0.9278. The graph is produced using data from the RIA method.
Figure 11 shows the correlation of (asialo-, monosialo- and disialo- transferrin) with disialotransferrin, determined in % transferrin. The squared correlation coefficient was 0.9906. The graph is produced using data from the RIA method.
CONCLUSION
From these Examples 8 and 9 it can be seen that there is a correlation between asialo and mono- & di-sialotransferrin, as well as between asialo- and disialo- transferrin and between either asialo- or disialo- transferrin and CDT (asialo- + monosialo- +
disialo- transferrin). Important to notice is that the monosialo fraction is small compared to disialo and will therefore only give a small contribution in the correlation study. This indicates that both asialo and disialo isoforms of transferrin can be used as parameters for monitoring alcoholic diseases. Trisialo was not seen to give any correlation to asialo and is probably not a good marker. Hence the clinical value of the assay according to the invention has been demonstrated, and combinations of transferrin variants suitable for use in producing the algorithm according to the invention have been found.
Furthermore, Example 9 illustrates that complete separation of the disialo transferrin is not required because the presence of the monosialo-variant in the determined or separated fraction is not harmful to the correlation results.
Serum transferrin is a glycoprotein with a molecular weight of about 80 kD which comprises a single polypeptide chain with two N-linked polysaccharide chains. These polysaccharide chains are branched and each chain may terminate in either two or three antennae, each with terminal sialic acid residues.
Wong and Regoeczi, in Int. J. Peptide Res. (1977) x:241-248, reported that human transferrin was naturally heterogeneous, occurring in variant forms with different levels of sialylation. Such variants are generally believed to be the hexasialo, pentasialo, tetrasialo, trisialo, disialo, monosialo and asialo transferrins.
Recently, it has been reported that the levels of monosialotransferrin are very low (see Landberg et al.
(1995) Biochem. Biophys. Res. Comm. 2,10(2):267-274 and Jochen et al. Biochemica et Biophysica Acta (1998) 1380:93-101).
The asialo, monosialo and disialo variants are referred to herein as carbohydrate-deficient transferrin or CDT. The asialo variant, now known to be completely devoid of carbohydrate chains, is referred to herein as carbohydrate free transferrin or CFT.
In the normal healthy individual, the tetrasialo variant appears to predominate; however it has been reported that the asialo, monosialo, disialo and, to some degree the trisialo variants occur in elevated levels in the blood of alcoholics (see van Eijk et al.
(1983) Clin. Chim. Acta ,;32:167-171, Stibler (1991) Clin. Chim. ,x:2029-2037 and Stibler et al. in "Carbohydrate-deficient transferrin (CDT) in serum as a marker of high alcohol consumption", Advances in the Biosciences, (Ed Nordmann et al), Pergamon, 1988, Vol.
71, pages 353-357).
CDT has been shown to be an effective marker for alcohol consumption, in particular for detecting and monitoring chronic alcohol consumption, and unlike conventional tests (e.g. quantitation of y-glutamyltransferase or measurement of mean corpuscular volume) can bemused to screen for heavy alcohol intake in patients with liver disease.
Recognition of the fact that the CDT profile of alcohol abusers differs from that of abstainers or normal users, combined with the identification of the relative amounts of each CDT isoform, e.g. on the basis of the differences in the isoelectric point (pI) or charge of the different transferrin molecules, has led to the development of several diagnostic assays for CDT.
These are described in the patent and scientific literature, for example in US-A-4626355, WO 91/19983, WO 96/26444, Heil et al. (1994) Anaesthetist 43:447-453, and Dumon et al. (1996) Clin. Biochem. x(6):549-553).
More recent studies (for example by Landberg et al.
(1995) Biochem. Biophys. Res. Comm. 210(2): 267-274), have shown, by releasing the N-glycans from each isoform of transferrin and analysing them by high-pH anion exchange chromatography, that the existence of disialo and asialo-transferrins correlates with the loss of one or both of the entire carbohydrate chains respectively from the transferrin polypeptide. This is confirmed by Jochen et al. in Biochemica et Biophysica Acta (1998) 1380:93-101.
Based on the discovery that the presence of transferrin isoforms which are completely devoid of carbohydrate (hereinafter referred to as carbohydrate free transferrin or CFT) is a strong indicator of WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 alcoholism in the absence of any knowledge of the prevalence of any other CDT variants (i.e. monosialo, disialo or trisialotransferrin variants), the Applicants developed and described in WO 99/00672 an assay for the determination of CFT as a means of assessing alcoholism.
The assay is robust, simple and quick to perform, and readily amenable to automation or compatible with existing routine clinical diagnostic laboratory procedures. This is achieved by separating the carbohydrate-containing transferrins from a sample by contacting the'sample with a carbohydrate-binding ligand, e.g. a lectin, and detecting and measuring the carbohydrate-free transferrin contained in the separated, non-binding fraction.
The accuracy and thus the clinical value of each of the assay procedures mentioned above is dependent on an efficient method of separation of the individual transferrin isoforms or combination of such isoforms.
As a result, relatively complex, expensive separation procedures are necessary to obtain an accurate diagnosis.
In particular, the pI or charge based methods of the prior art are primarily centred on procedures involving ion exchange chromatography. The difference in pI between the different transferrin variants is very narrow, down to 1/10 of a pH unit and therefore to effect separation of CDT variants, a very good separation is required. In the case of ion exchange chromatography, this constraint effectively means that a column format must be used; batch filtration-based ion exchange procedures do not provide a sufficient separation or resolution. Column formats are however less preferred in clinical chemistry or diagnostic procedures, due to their time consuming and labour intensive operation, problems of storage and transport, incompatibility with commonly-used systems etc.
Due to the low levels of CFT generally found in serum samples (even in alcoholics only about to of the transferrin molecules are asialotransferrin or CFT), an efficient separation is similarly required to effect separation of CFT from any carbohydrate containing transferrins in the assay described in WO 99/00672.
There is therefore a need for an alternative assay method in which incomplete or partial separation of transferrin variants, e.g. one or more CDT variants, can be tolerated without affecting the clinical value of the assay. The present invention seeks to address this need.
Martensson et al. (Alcoholism: Clin. and Exp.
Research (1997) x(9):1710-1715) describe a clinical study of transferrin variant concentrations by means of ion exchange HPLC in combination with RIA analysis of the transferrin content of the HPLC fractions eluted.
Higher clinical sensitivity was found for measurement of disialotransferrin alone and with the sum of asialo-, monosialo- and disialo-transferrins compared to asialo-and monosialo-transferrins alone. A much lower clinical sensitivity was found with trisialotransferrins.
M~rtensson et al. focus on the differences in clinical signals between the different transferrin variants with a view to selecting the best transferrin variant or combination of variants. No correlation figures for the different variants are reported.
In June 1999, Professor Jan-Olof Jeppson working at the University of Malmo, Sweden reported a weak correlation (correlation coefficient RZ - 0.63) between asialo and disialo-transferrins using the procedure of ion exchange chromatography and spectrophotometric detection at 470 nm as described in WO 95/04932.
Contrary to the findings of Jeppson, we have now surprisingly found a high level of correlation between the asialo- and disialo-transferrin contents of serum samples. This has led to the development of an improved assay procedure for determining the content of any desired transferrin variant or combination of transferrin variants, in particular asialo- or disialo-transferrin or CDT, in a body fluid sample in which a less efficient separation of transferrin variants can be tolerated. This allows much simpler separation methods to be used for the assessment of alcohol consumption.
Thus, according to one aspect, the present invention provides a method of producing an algorithm for determining the content of a transferrin variant or combination of transferrin variants, preferably a CDT
variant or combination of CDT variants, in a sample of body fluid, said method comprising:
(a) obtaining at least two solutions each having known contents of asialo- (A1, A2, A3, etc.) and disialo-transferrins (D1, D2, D3, etc.);
(b) subjecting each of said solutions to a separation method whereby to separate fractions substantially free from tri- and higher sialylated transferrins (c) determining the total transferrin variant content (T1, T2, T3, etc.) of said fractions; and id) producing an algorithm capable of determining the content of any transferrin variant or combination of transferrin variants, preferably a CDT variant or combination of CDT variants, in any given sample of body fluid subjected to said separation method.
As mentioned further below, however, the purpose of the separation step used in the methods herein described is essentially to remove all or substantially all tri-, tetra-, penta- and hexa-sialotransferrins (non-target variants) from the sample, allowing the remaining target variants (asialo-, monosialo- and disialotransferrins) to be determined Thus viewed from a broader aspect, the invention provides a method of producing an algorithm for WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 determining the content of a transferrin variant or combination of transferrin variants, preferably a CDT
variant or combination of CDT variants, in a sample of body fluid, said method comprising:
(a) obtaining at least two solutions each having known contents of asialo- (Al, A2, A3, etc.) and disialo-transferrins (D1, D2, D3, etc.);
(b) determining the content in each of said solutions of a transferrin variant or combination of transferrin variants in fractions substantially free from tri- and higher sialylated transferrins;
(c) determining the total transferrin variant content (T1, T2, T3, etc.) of said fractions; and (d) producing an algorithm capable of determining the content of any transferrin variant or combination of transferrin variants, preferably a CDT variant or combination of CDT variants, in any given sample of body fluid subjected to said determination step b) .
In general, the algorithm produced in accordance with the invention will be one capable of determining the actual content or amount of asialo- or disialo-transferrin, or the actual content or amount of asialo-, monosialo- and disialo-transferrins (i.e. CDT) in any sample of body fluid.
As will be apparent, the algorithm will be specific to a given determination step or separation method carried out under specific conditions. Preferably, the algorithm for use in the invention will be based upon a quantitation of each of the isoforms of transferrin having zero, one or two sialic acid residues per molecule. Most preferably, the algorithm may be produced on the basis of the high correlation between -the content of asialo- (CFT) and disialo-transferrins in a sample, preferably also the low level of monosialotransferrin.
Based on the correlation determined between asialo-and disialotransferrins, the invention essentially provides a method of producing a calibration curve specific to any given determination step or separation method and which may be used to calculate the content or amount of any transferrin variant or variants in any given sample of body fluid. The correlation coefficient between asialo,'- and disialotransferrin may be determined using mathematical techniques and correlations standard in the art, e.g. least squares analysis.
More specifically, the algorithm produced in accordance with the invention may be defined by at least one of the following equations:
A = (T-d.b) / (c + d.a) D = b + a (T-d.b) / (c + d.a) CDT = A + D = b + (a + 1) (T-d.b) / (c + d. a) (wherein T represents the determined total transferrin content in the determined fraction (or in the separated fraction following separation);
A represents the actual asialotransferrin content in the sample;
D represents the actual disialotransferrin content in the sample;
CDT represents the actual total content of asialo-, monosialo- and disialotransferrins in the sample;
a and b are constants defining the correlation between A
and D in any serum sample; and c and d are constants specific to the determination step or separation method).
- g _ In a further aspect the invention provides a method for determining the content of a transferrin variant or combination of transferrin variants, preferably a CDT
variant or combination of CDT variants, in a body fluid for use in the assessment of alcohol consumption, said method comprising:
(a) subjecting a sample of said body fluid to a separation method capable of separating a fraction substantially free from tri- and higher sialylated transferrins;
(b) determining the total transferrin variant content in said fraction; and (c) determining the content of any transferrin variant or combination of transferrin variants, preferably the content of any CDT variant or combination of CDT variants, in said sample using an algorithm obtained in accordance with a method as herein described.
However, the separation step need not be provided if an alternative way to determine the target variants (asialo-, monosialo- and disialo- transferrins) is available. Furthermore, it is also possible to determine the content of only a portion of the target variants, so long as the portion of target variants is constant and reproducible.
Thus viewed from a broader aspect, the invention provides a method for determining the content of a transferrin variant or combination of transferrin variants, preferably a CDT variant or combination of CDT
variants, in a body fluid for use in the assessment of alcohol consumption, said method comprising:
(a) determining the content in a sample of said body WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 g -fluid of a transferrin variant or combination of transferrin variants in a fraction substantially free from tri- and higher sialylated transferrins;
(b) determining the total transferrin variant content in said fraction; and (c) determining the content of any transferrin variant or combination of transferrin variants, preferably the content of any CDT variant or combination of CDT variaf-~ts, in said sample using an algorithm obtained in accordance with a method as herein described.
In the assay in accordance with the invention, the measured transferrin variant content can be used to determine the actual levels of any transferrin variant or variants in the sample on the basis of a calibration derived from the correlation discovered for the levels of asialo- and disialotransferrins.
As will be generally understood, the determination step or separation method used in performing the assay in accordance with the invention will be the same as that used to produce the algorithm or calibration curve, such determination steps or separation methods being performed under the same set of conditions.
As used herein, the terms "determining" or "assessing" include both quantitation in the sense of obtaining an absolute value for the amount or concentration of transferrin variants) in the sample, and also semi-quantitative and qualitative assessments or determinations. An index, ratio, percentage or similar indication of the level or amount of transferrin variant(s), for example relative to total transferrin (i.e. all transferrin variants) may be obtained.
The assay method of the invention provides a convenient method for the determination of alcohol consumption by assaying the level of a transferrin variant or combination of transferrin variants in a body fluid, preferably a blood-derived body fluid, and may particularly find utility in the diagnosis and monitoring of alcoholism or alcohol abuse. Both asialo-and disialotransferrins, as well as a combination of asialo-, monosialo- and disialotransferrins have previously been shown to be a good indicator or marker for alcoholism or alcohol abuse, and by assaying the content of any of these in samples of body fluid, a distinction may be found between alcoholics and alcohol abusers and noh-alcohol abusers or social drinkers.
The body fluid used in the assay method of the invention may be any transferrin-containing body fluid for example, synovial fluid, amniotic fluid or cerebrospinal fluid, but will generally be blood or a blood derived sample. When this is the case, the sample used for analysis will preferably be cell-free and hence, either serum or plasma may be used. The sample may be treated prior to being used in the assay method of the invention, for example, it may be diluted by adding a buffer or other aqueous medium.
The purpose of the separation step used in the methods herein described is essentially to remove all.or substantially all tri-, tetra-, penta- and hexa-,. 25 sialotransferrins (non-target variants) from the sample, allowing the remaining target variants (asialo-, monosialo- and disialotransferrins) to be determined.
Where a determination step is referred to in relation to the methods herein described, this may in a preferred embodiment be a separation step. However, it may also be desirable in some circumstances to directly determine the content of the target variants) in a fraction of the sample without physically separating the fractions out.
For example, various immunoassay techniques are available which could allow detection or quantitation of target transferrin variants) in a body fluid. The antibody described in EP 0 605 627 is specific for a transferrin homolog found in alcoholics, or CDT, and therefore such an antibody may be used to determine the content of a target transferrin variant, or combination of transferrin variants. Provided that the antibody binds a constant fraction of the sample which is substantially free from tri- and higher sialylated transferrin it is possible to produce an algorithm in accordance with the invention.
Thus, in a particularly preferred aspect of the invention, the determination step is effectively a direct determination or direct measurement of the content of the target transferrin variants, or a combination of said target variants.
Transferrin preparations which may be regarded as substantially free from tri-, tetra-, penta- and hexa-sialotransferrins are those comprising less than 200, more preferably less than 10%, e.g. less than 5%
transferrin variants having three or more sialic acid residues per molecule.
Provided that the determination step or separation method is reproducible, separation of all asialo-, monosialo- and disialotransferrins or determination of the content of each is not required when carrying out the invention. Although possible to isolate all CDT
variants, clinically valuable results can be obtained by separating or determining only a fraction of these variants. What matters is that the separated or determined fraction of each variant is reproducible (i.e. essentially constant) for a given determination step under given conditions, or for a given separation method and given separation conditions. Quantitation (in the sense of obtaining an absolute value) of any or all of the CDT variants is not essential in carrying out the methods herein described.
Following separation (where separation is carried out), or in the case of a determination step, the fraction containing the desired target variants will typically comprise at least 60%, preferably at least 70 to 80%, e.g. 90 to 95% of the asialo- and monosialo-transferrin variants present in the sample prior to separation or determination. The disialotransferrin content of the separated or determined fraction will generally be at least 20%, preferably up to 60 to 700, particularly preferably 20 to 500, e.g. about 300 of the total disialotransferrin content of the sample prior to separation or determination.
In the separated or determined fraction containing the desired target variants, at least 40%, preferably at least 60%, e.g'. at least 70 to 80% of the transferrin molecules will carry a carbohydrate chain or a residue thereof. The separated or determined fraction will typically comprise less than 200, e.g. 10 to 150 asialotransferrin (or CFT), less than 5%
monosialotransferrin and 70 to 80%, e.g. about 750 disialotransferrin. An amount of trisialotransferrin, typically up to 20%, preferably up to 100, e.g. up to 50, can be tolerated without affecting the clinical value of the assay.
Thus for example the fraction to be determined or separated could consist essentially of asialotransferrin or it could could consist essentially of disialotransferrin, but in either case some or all of monosialotransferrin (and/or trisialotransferrin) could be present in the same fraction. Because the amount of monosialo (or trisialo-) transferrin is low relative to the amounts of asialo- and disialo- transferrin, this will not interfere significantly with the results of an assay in accordance with the invention.
Conveniently, ion exchange chromatography may be used to remove all or substantially all of the tri- and higher sialylated transferrins in any of the methods herein described. Ion exchange as a means of separating the various isotransferrin components is well known, and is described for example in US-A-4626355, Heil et al.
(supra) and WO 96/26444. Advantageously, an anion exchange chromatography step may be used, with the chromatography conditions (e. g. pH and ion binding strength) selected to permit retention of the desired transferrin variants (e.g. hexa-, penta-, tetra- and tri-sialo transferrin, and optionally some or all of the disialo fraction) .
Appropriate conditions e.g. buffering-capacity of the resin, sample/equilibration/elution buffer pH and/or ionic strength can readily be determined according to techniques known in the art, and according to the separation desired to be achieved. As is known in the art, prior to ion exchange, the sample may be treated with iron-containing buffer to saturate the iron-binding sites in the transferrin molecules in the sample.
Conveniently, according to techniques known in the art, chloride may be used as the counterion in the ion exchange procedure in order to achieve the desired separation. Thus, appropriate amounts of chloride ion present in the chromatography procedure necessary to achieve retention of the desired transferrin variants may be determined by routine experiments, and may depend on the precise conditions, batch of chromatography medium etc. The procedure can be monitored by isoelectric focusing or HPLC analysis, again according to standard techniques known in the art.
The ion exchange chromatography step may be carried out in any convenient manner known in the art according to choice, e.g. in a batch or column format. Likewise, the conditions may be selected to achieve the separation (i.e. depletion or removal) in any desired manner, for example by retaining the isotransferrin variants it is desired to remove (i.e. target variants), or by pre-treating the sample by ion exchange such that the "undesired" (i.e. non-target) variants do not absorb to the medium, and the remainder of the sample is separated and then eluted from the ion exchange medium.
Advantageously, the chromatography conditions are set to permit retention of the "undesired" transferrin variants.
As exemplary of ion exchange conditions which may be used, mention may be made of Whatman QASL anion exchange resin buffered at pH 6.3, which may be used to bind the trisialo and higher sialylated transferrins.
Alternatively, separation of target and non-target variants may be achieved using a binding ligand capable of binding selectively either to the target or non-target transferrin variants. Any binding ligand which has an affinity for the target or non-target transferrin variants may thus be used to separate the target variants from bther transferrin variants in the sample.
Preferably, the binding ligand may be a carbohydrate-binding ligand such as a lectin or mixture of lectins, e.g. as described in WO 99/00672. As noted in this earlier patent, 100% separation of CFT cannot always be achieved in practice. It has now been found that transferrin variants having a low carbohydrate content, in particular monosialo- and disialo-transferrins, bind with a low affinity to carbohydrate-binding ligands, such as lectins. In particular, it has been found that such variants bind to carbohydrate-binding ligands with a lower affinity than the transferrin variants having a higher or high carbohydrate content (i.e. the higher sialylated transferrins, e.g. tri-, tetra- and pentasialotransferrins). Separation methods using carbohydrate-binding ligands, for example lectins, are thus effective to remove higher sialylated transferrins (e. g. tri-, tetra-, penta- and hexa-sialotransferrins), but are generally less effective in removing lower sialylated transferrins (e.g. mono- and di-sialotransferrins). The result is an incomplete separation of CFT in which the separated "non-binding"
fraction will typically contain some or all of the mono-and disialotransferrins in addition to asialotransferrin (CFT). As noted previously, the assay method in accordance with the invention can tolerate incomplete separation of transferrin variants without compromising the clinical value of the assay.
WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 When the body fluid comprising transferrin variants is contacted with the carbohydrate-binding ligands, substantially all of the higher sialylated variants (tri-, tetra-, penta- and hexa-sialotransferrins) with carbohydrate side chains or remnants thereof are retained by the carbohydrate-binding ligands. The unbound fraction containing CFT, mono- and disialotransferrins may then be separated from the other variants and collected by any suitable means.
In a particularly preferred embodiment of the invention the separation method comprises the steps of contacting a sample of body fluid with a carbohydrate-binding ligand, e.g. a lectin or mixture of lectins, followed by separation of a fraction not binding to said ligand. The total amount of transferrin in the non-binding fraction may be determined directly by measuring the transferrin not bound by the carbohydrate binding ligand. Alternatively it may be determined indirectly by determining the amount of transferrin bound to the carbohydrate binding ligand and subtracting this from the total amount of transferrin present in the sample.
Generally, the direct approach is preferred.
Any carbohydrate-binding ligand or any combination thereof may be used to separate the target variants from other transferrin variants. This includes any ligand capable of binding to any carbohydrate or oligosaccharide or sugar structures. One or more carbohydrate-binding ligands may be used in the method of the invention. Where more than one ligand is being used, these may be used together or they may be used individually, for example, sequentially. The functional requirement of the carbohydrate binding ligand(s), rendering them suitable for use in the assay method of the present invention, is that they be capable of separating CDT from other transferrin variants.
Generally, the carbohydrate-binding ligand will be a protein, and very many such carbohydrate-binding proteins are known in the art and are widely described in the literature. The carbohydrate-binding protein may, for example, be an antibody, either polyclonal or monoclonal, or may be an antibody fragment for example F (ab) , F (ab' ) z or F (v) fragments . The antibodies or antibody fragments may be monovalent or divalent and they may be produced by hybridoma technology or be of synthetic origin, via recombinant DNA technology or chemical synthesis. Single chain antibodies could for example be used. The antibody may be directed or raised against any of the carbohydrate components or structures making up the Carbohydrate chains of glycosylated transferrin variants. Thus, for example, an antibody reactive with or selective for sialic acid residues might be used. Such an antibody is used in the Sialic Acid Deficient Enzyme Immunoassay (SDT-EIA) available from Medichem, Stuttgart, Germany and described in WO
97/19355.
More preferably, the carbohydrate-binding protein may be a lectin, used singularly or in combination with other lectins or with other types of carbohydrate-binding proteins, for example, antibodies. Any lectin known in the art may be used in the assay method of the invention and it may be of plant, animal, microbiological or any other origin. The literature is replete with references to different lectins which might be used, and many may be obtained commercially, for example, from Sigma.
Thus, included within the general term "lectin" as used herein, in addition to the classical plant lectins such as Concanavalin A (Con A), are carbohydrate binding proteins from microorganisms (for example, viral haemagglutinins) and higher organisms, including for example, invertebrates and mammals. Such mammalian carbohydrate binding proteins include selecting and other mammalian lectins or cell adhesion molecules (see for example Varki (1992) Current Opinion in Cell Biology 4:257-266).
Examples of suitable lectins are RCA-I (Ricinus communis agglutinin) which binds terminal galactose (Kornfeld et al. (1981) J. Biol. Chem. 256:6633) or Con-A (Concanavalin A), which is known to bind asparagine-linked oligosaccharides high in mannose. Other possibilities are Crotalaria juncea lectin which binds galactose residues (Ersson (1977) Biochim. Biophys. Acta 494:51-60), Wheatgerm agglutinin or Limulus polyphenus lectin which bind sialic acid (Mandal and Mandal (1990) Experientia 46:433-441) or Sambucus nigra agglutinin L
which binds NeuSAc/(«2-6)Gal/GalNAc (Shibuya et al.
(1987) J. Biol,'. Chem. 262:1596). As an example of a lectin derived from a micro-organism, a sialic acid specific lectin has recently been purified from the gut dwelling organism Helicobacter pylori (Lelwala-Guruge et al. (1993) APMIS 101:695-702).
Lectins of varying selectivity and specificity are known. Whereas some lectins may bind to a single sugar residue in a particular location on an oligosaccharide chain, for example RCA-I (from Ricinus communis) binds only to terminal galactose residues, some may bind to complex oligosaccharide determinants for example Sambucus nigra L which binds NeuSAc/(«2-6)Gal/GalNAc.
All are within the scope of the present invention.
Sialic acid binding lectins and other proteins represent a class of carbohydrate binding proteins of particular utility in the present invention (see the following, for example, for lists of suitable lectins and their sources: Mandal and Mandal(1990) Experientia 46:433-441); Zeng (1992) Z. Naturforsch, 47c:641-653 and Reuter and Schauer in Methods in Enzymology, Vol. 230, Chapter 10 at pages 196-198).
Particular mention may be made in this regard of Sambucus nigra L. Lectin, Sambucus sielbodiana lectin wheatgerm agglutinin, Maackia amurensis lectin, and E.
coli K99 lectin. S. nigra L. lectin is particularly effective when used on its own, although it may equally effectively be used in combination with other lectins eg. ConA.
WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 Some particular combinations of carbohydrate binding ligands useful for performance of the present invention are lectins from Helicobacter pylori and Ricinus communis; lectins from Ricinus communis and Sambuccus nigra; lectins from Crotalaria junctae and Sambuccus nigra; lectins from Crotalaria junctae and Helicobacter pylori and lectins from Ricinus communis and anti-sialic acid antibodies. The most preferred of the combinations are those which incorporate galactose-binding and sialic acid-binding ligands.
Followin~'the binding steps) a fraction may conveniently be collected which does not bind and which contains the CDT. Collection may be by any suitable means, for example, precipitation, centrifugation, filtration, chromatographic methods etc. Where different carbohydrate-binding ligands are used individually, different separation/collection formats may be used for each individual binding step.
Precipitation of carbohydrate-containing moieties in the sample may be achieved using lectins having known "precipitation" properties i.e. lectins capable of inducing precipitation of the moieties to which they bind. Combinations of lectins may advantageously be used for such a precipitation procedure, since differing lectin specificities increase the number of available binding sites. The non-binding (CDT) fraction may then readily be collected, for example by centrifugation or filtration to separate the precipitate.
In alternative embodiments, the carbohydrate binding ligand(s) may conveniently be immobilised to facilitate the separation and collection of the non-binding fraction. It is well known in the art to immobilise carbohydrate-binding ligands such as lectins for separation purposes, for example, in chromatographic columns, and any lectin affinity chromatography method known in the art could for example be used (see for example, Cummings (1994) Methods in Enzymology 230:66-86) .
WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 The carbohydrate-binding ligands may be immobilised by binding or coupling to any of the well known solid supports or matrices which are currently widely used or proposed for immobilisation or separation etc. These may take the form of particles, sheets, gels, filters, membranes, fibres or capillaries or microtitre strips, tubes or plates or wells etc and conveniently may be made of glass, silica, latex or a polymeric material.
Techniques for binding the ligand to the solid support are also extremely well known and widely described in the literature'. For example, the carbohydrate-binding ligands used may conveniently be coupled covalently to CNBr-activated Sepharose or N-hydroxysuccinimide-activated supports, optionally in the presence of low molecular weight haptens to protect the carbohydrate binding sites on the ligand. Other coupling methods for proteins are also well known in the art.
Batch separations using immobilised carbohydrate-binding ligands may be performed using a range of different formats which are known in the art.
In a different embodiment, although this is less preferred, the immobilised carbohydrate-binding ligands may be packed or arranged into a column. The body fluid comprising transferrin may be applied to the column and the transferrin variants therein contacted with the carbohydrate-binding ligands. The unbound fraction comprising CDT is separated from the bound fraction and collected.
The shape and geometry of such a column may vary depending upon the carbohydrate-binding ligands used.
For example, if lectins are used as the carbohydrate-binding ligands, at low lectin concentrations a long, thin column of immobilized lectins is preferred. At high lectin concentrations, column geometry is less crucial.
Columns may be constructed using any method known in the art. If lectins are to be used as the carbohydrate-binding ligands, the columns may be constructed in either glass tubes or preferably in disposable plastic pipettes of any desired capacity.
Smaller volumes may however be preferred due to economic considerations. Columns are preferably stored at around 4°C prior to use.
The column may be flushed through with an eluant to allow or facilitate collection of the unbound fraction, in which case the eluant should preferably be administered using a calibrated micropipette to ensure the correct volume is administered. The volume administered i~ preferably within 30 of the desired (i.e. calibration) volume, more preferably, within 1 or 2%. Since the rate of binding to oligosaccharides is comparatively slow, especially with plant lectins, it is preferable that slow flow rates are employed to maximise lectin/carbohydrate interactions. The eluant will generally be at a temperature within 5°C of the desired (calibration) value, e.g. 25°C, and more preferably within 1°C.
When using combinations of carbohydrate-binding ligands in a column format, either sequential columns using different ligands may be used or different ligands may be used in the same column material, either as a mixture or in a column comprising different layers, each layer having a different ligand.
In an alternative embodiment, the carbohydrate-binding ligand may be immobilised on a particulate solid phase, for example, latex, silica or polymer beads. To aid manipulation and separation, magnetic beads may be used. The term "magnetic" as used herein means that the support is capable of having a magnetic moment imparted to it when placed in a magnetic field. In other words, a support comprising magnetic particles may readily be removed by magnetic aggregation, which provides a quick, simple and efficient way of separating the fractions following the carbohydrate binding step.
Thus, the magnetic particles with non-target variant moieties attached may be removed onto a suitable surface by application of a magnetic field, for example, using a permanent magnet. It is usually sufficient to apply a magnet to the side of the vessel containing the sample mixture to aggregate the particles to the wall of the vessel and to collect the remainder of the sample, which will comprise the "non-binding, CDT-containing fraction" which may be returned for subsequent analysis.
Especially preferred are superparamagnetic particles, which include for example those described by Sintef in EP-A-106873, as magnetic aggregation and clumping of the particles during the reaction can be avoided. Magnetic particles are commercially available from a number of sources, including for example, Advanced Magnetics Inc., (USA), Amersham (UK), Bang Particles (USA), and Dynal AS (Oslo, Norway).
Functionalised coated particles for use in the present invention may be prepared by modification of the beads, for example according to US patents 4,336,173, 4,459,378 and 4,654,267. Thus, beads, or other supports, may be prepared having different types of functionalised surface, for attachment of a desired carbohydrate-binding ligand.
Separations based on centrifugation and/or filtration are convenient. In a preferred embodiment a centrifuge tube (e. g. Eppendorf tube) and "filter cup"
format may be used, and such formats are readily commercially available, for example from Millepore.
Thus the sample and carbohydrate-binding ligand may be added to the cup in the tube and allowed to bind. The tube (and cup) is then spun, and the non-binding supernatant collects in the tube. The carbohydrate-binding ligand may be such as to induce precipitation of the bound carbohydrate moieties or it may be immobilised, for example as a slurry e.g. a gel or on particles. In either case, the bound carbohydrate binding fraction is retained in the cup.
As a variation of such a "tube and cup"
arrangement, the cup may be provided with one or more WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 "discs" or filters which carry immobilised carbohydrate-binding ligands.
Following the determination step or the separation step, the total content of transferrin in the separated fraction is determined. This may be done by any standard procedure known in the art for assay of transferrin, for example, by any standard immunoassay technique, e.g. an ELISA or radio-immunoassay technique.
Methods for determining transferrins are described for example in US-A-4,626,355 (Joustra).
An example of an ELISA method could include a sandwich assay in which an immobilised antibody specific for transferrin variants which are substantially free from tri- and higher sialylated transferrins is contacted with the sample, and then enzyme-labelled anti-transferrin antibodies (available from Dako AS, Denmark) are used to detect the bound transferrin. A
preferred such immobilised antibody is the antibody described in EP 0 605 627 and in such a case it will be appreciated that the separated fraction will be the fraction of antibody-bound transferrin and that the enzyme signal will be proportional to the total transferrin content of that fraction, allowing the total transferrin to be determined in that fraction.
Many commercial assays for transferrin are available and have been described in the literature.
For example an RID (radio immuno diffusion) assay based on the method of Mancini is available from Hoechst (see Mancini et al. Immunochemistry, 2:235-254 (1965)). A
rocket immuno electrophoresis method is described by Laurell in Scand. J. Clin. Lab. Invest. 29 (Suppl. 124):
21-37 (1972). Particular mention may also be made of the particle-based immunoassay method of Miiller et al.
in Lab. Med. 1:278 (1991). This is a very sensitive technique, based on an enhanced turbidometric method which uses a turbidometric signal but is more sensitive than traditional turbidometric methods.
For either turbidimetric or nephelometric transferrin determination, opacity will generally be generated by contacting the separated fraction or an aliquot thereof with an anti-transferrin antibody or antibody fragment, e.g. a rabbit anti-human transferrin antibody such as is commercially available from Dako of Copenhagen, Denmark. The Dako antibodies are specific to transferrin and show no cross reactions with other blood proteins that may be present in the eluate. The quantity of antibody used should of course be optimised against transferrin containing standard samples as opacification irises from the hook effect whereby multiple transferrin binding generates the opacification centres.
In the case of the "tube and cup" embodiment described above for example, the anti-transferrin antibodies may simply be added to the tube after centrifugation.
As in routine turbidimetric and nephelometric assays, a polymeric opacification enhancer, such as polyethyleneglycol, is preferably also added to the eluate.
In determining transferrin content using such measuring techniques, a kinetic reading mode may of course be used.
Before the nephelometric or turbidimetric determination is made, the fraction, antibody and enhancer may be incubated for a short period, e.g. 5 minutes to an hour for end-point measurements, preferably about 10 minutes.
The light used in the determination of opacification should have an appropriate wavelength. In this regard we have found that use of a 405 nm filter, or more preferably a 340 nm filter, yields particularly good results.
Where a separation method is to be avoided and a direct determination step is instead to be used, a suitable method is to take advantage of a proximity interaction technique. Most preferably, in this embodiment of the invention, a specific binding partner for the target transferrin variants is utilised in a proximity assay to obtain a direct determination of the amount of the target variants (asialo-, monosialo- and disialotransferrins) in the fractions) to be determined. An example of a suitable specific binding partner is the anti-transferrin antibody which reacts selectively with transferrin homologs found in alcoholics but not in non-alcoholics, as disclosed in EP 0 605 627.
Another means of producing a suitable specific binding partner with appropriate specificity for the target transferrin variants to be determined is to raise an antibody against a peptide immunogen which mimicks the N-glycan binding site on the transferrin molecule, for example if the peptide sequence corresponds to the amino acid sequence of human transferrin. The amino acid sequence of human transferrin is known and is published for example in Yang et al., Proc. Natl. Acad.
Sci. 81: 2752-2756 (1984) or accession number PO 2787 Swiss Prot database.
Examples of suitable techniques which allow for detection of a signal due to proximity interaction between molecules are described in the literature. Such techniques include fluorescence polarisation immunoassay technology (FPIA), fluorescence quenching techniques, proximity scintillation assays and EMIT technology.
FPIA techniques are described for example in Dandliker et al., Immunochemistry 7: 799-828, (1970), and in Wei et al., Anal. Chem. 65: 3372-3377 (1993)).
Fluorescence quenching techniques are described for example in US-A-3,996,345 of Ullmann et al., in which one binding partner carries a fluorescent residue and the other carries a quencher.
Proximity scintillation assays are described for example in US-A-4,568,649 and EP 0 154 734 of Bertoglio-Matte. In these assays, one of the binding partners emits a short-range energy-rich radioactive signal, WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 typically emitting (3-rays, and when proximity interaction with the other binding partner occurs, a fluorophore linked to the second binding partner is excited by the radioactive energy and a fluorescent signal is generated.
A further, more complex type of fluorescent proximity assay was described in US-A-5,763,189 of Buechler et al. which is based upon the measurement of Stokes shift (difference in wavelength of light which is emitted as compared to the excitation wavelength).
The EMIT technology is described for example in US-A-3,852,157 of Rubenstein et al. and this technique relies on competition between analyte molecules and enzyme-labelled analyte analogs (the "reactant") for a receptor (the "specific binding partner") in the assay solution.
Further signal detection techniques include turbidimetry and nephelometry as described hereinbefore in relation to separation methods.
In general, besides the sample under evaluation, calibration samples with known transferrin contents will also be assessed in the performance of the assay method of the invention. Such determinations can be used to plot a calibration curve from which the transferrin content of the sample under evaluation may be determined. Preferably calibration samples having transferrin contents of up to 0.05 mg/ml (e. g. 0.002, 0.01, 0.02 and 0.03 mg/ml) will be used.
In the assay method of the invention the total transferrin content of the determined or separated fraction containing the target variants will preferably be determined. Using an algorithm as herein described this can then be used to determine with a high degree of accuracy the content of asialo- or disialo-transferrin, or CDT content of the sample. The content of asialo- or disialo-transferrin or CDT may be determined as a percentage of total transferrin. This may be a more precise marker for alcohol consumption than total transferrin, and a threshold value, for example lo, may be set. Alternatively, the presence of any transferrin variant may be assessed as an actual concentration (i.e.
a mass per unit volume).
Viewed from a further aspect, the invention provides a kit for a diagnostic assay according to the invention, said kit comprising:
means for subjecting a sample of body fluid to a determination step or separation method capable of producing or determining the content of a fraction substantially ;free from tri- and higher sialylated transferrins;
means for the detection of transferrin; and means for determining the content of any transferrin variant or combination of transferrin variants in a sample of body fluid subjected to said separation method or determination step.
Conveniently, the kit may also comprise a transferrin standard or standards for reference. Thus, in one preferred embodiment, the kit of the invention may comprise:
at least two transferrin solutions having known asialo- and disialo- concentrations;
means for subjecting a sample of body fluid to a separation method or determination step capable of producing or determining the content of a fraction substantially free from tri- and higher sialylated transferrins;
means for the detection of transferrin; and means for determining the content of any transferrin variant or combination of transferrin variants in a sample of body fluid subjected to said separation method or said determination step.
The invention will now be illustrated by the following non-limiting Examples and the accompanying figures in which:
Figure 1 shows the high correlation (R2 - 0.9153) between the concentration of asialo- and disialotransferrin variants in a serum sample determined in accordance with the method of Example 1.
Figure 2 shows the high correlation (R2 - 0.9336) between the o of asialo- and disialotransferrin variants in a serum sample determined in accordance with the method of Example 1.
Figure 3 shows separation of transferrin isoforms by HPLC. ' Figures 4A and 4B show correlation of asialo vs monosialo, in A determined in o transferrin, in B
determined in ~.g/mL.
Figures 5A and 5B show correlation of asialo vs disialo, in A determined in o transferrin, in B
determined in ~.g/mL.
Figure 6 shows the correlation of (asialo-, monosialo- and disialo- transferrin) with asialo transferrin, determined in mass units (~,g/mL) .
Figure 7 shows the correlation of (asialo-, monosialo- and disialo- transferrin) with disialotransferrin, determined in mass units (~g/mL).
Figure 8 shows the correlation of asialo transferrin with (monosialo- and disialo-) transferrin, determined in o transferrin.
Figure 9 shows the correlation of asialo- with trisialo- transferrin, determined in o transferrin.
Figure 10 shows the correlation of (asialo-, monosialo- and disialo-transferrin) with asialotransferrin, determined in o transferrin.
Figure 11 shows the correlation of (asialo-, monosialo- and disialo- transferrin) with disialotransferrin; determined in o transferrin.
Example 1 Reference method - Determination of asialo-, monosialo-, disialo- and trisialo-transferrin content of serum samples The transferrin variant content of lipid stripped, iron treated serum samples was analysed using a combined HPLC-RIA method. The levels of the different sialic acid isoforms were determined first by separation on HPLC with an ibn exchange column. The asialo-, monosialo-, disialo- and trisialotransferrin fractions were collected in separate tubes. The higher isoforms, tetrasialo-, pentasialo- and hexasialo-transferrins, were collected in a separate tube. The a of the different fractions was first determined using HPLC, then the concentration of the fractions was determined on a gamma counter using an immunoassay procedure as outlined below.
Reagents Dextransulfate sodium salt Calcium chloride dihydrate Ferric chloride, FeC13.6H20, min. 99.0%
Acetic acid > 990 2-propanol Nitrilotriacetic acid trisodium salt monohydrate >980 BisTris (bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane) > 99.50 NaCl p.a.
2 M NaCl, p.a.
2 M NaOH, p.a.
5o Acetic acid 75o Acetic acid Mobile phase A: 20mM BisTris buffer, pH 6.5 Mobile phase B: 20mM BisTris buffer, pH 6.5, 0.5M NaCl Mobile phase C: 20mM BisTris buffer, pH 5.8 Mobile phase D: water Equipment - 2 ml syringe - 0.2 ~m filter for filtering serum samples (Gelman Acrodisc 13) - Column: Pharmacia Resource Q 1 mL, Code 17-1177-O1 or Pharmacia Source 15Q 1 mL
- Pre-column: HP Pre-column cartridge holder 4.Omm inner diameter, filled with HQ50 in slurry form - Transferrin, in solution and labelled with lzsI
- Anti-transferrin antibody (raised in rabbit) solution - Sheep anti-rabbit antibody (decanting solution) - Calibrators 0, 5, 20, 50, 100 and 300 U CDT/L
(1 U/L = 0.034 ~.g/ml) - Transferrin calibrators (based on Seronorm calibrated serum supplied by Sero AS, Norway, diluted in mobile phase A) 0, 0.05, 0.10, 0.15, 0.25, 0.50, 0.75, 1.0, 1.5, 2.5 ~,g/ml - HPLC Degaser: Degaser Mod. G1322A
- HPLC Pump: Quaternary pump Mod. G1311A
- HPLC Injector: Autosampler Mod. G1313A, modified with a 900,1 injector loop - HPLC Thermostat: Column thermostat Mod. G1316A, with a Reodyne multi-port - HPLC Detector: Diode Array Detector Mod. G1315A
Data Handling: HP ChemStation -- pH meter: Hanna Instruments 8417 - Centrifuge: Minifuge RF, Z-924 - Gamma counter: RIASTAR
- Fraction collector: GradiFRAC from Pharmacia HPLC
150.1 of each serum sample was added to 30.1 FeNTA (an aqueous solution containing 2.751 g/1 nitrilotriacetic trisodiummonohydrate and 2.703 g/1 FeC13.6H20 adjusted to a pH of 6.5 using 2M NaOH) and vortexed. The resulting solution was then added to 101 dextran sulfate (20 mg/ml) and 10.1 potassium chloride (147 mg/ml). The solution was cooled to 2-8°C for 30 mins. The sample was then centrifuged at 3800 rpm for 10 mins. 150,1 of the supernatant was pipetted out and added to 900.1 HPLC
buffer A. The sample was filtered on an acrodisc filter and then injected into the HPLC in an aliquot of 8001.
The transferriri isoforms were separated by an ion-chromatographic gradient method. The transferrin was selectively detected spectrophotometrically at 470 nm by the HP ChemStation.
Five fractions of the transferrin were collected from the HPLC into tubes in exact amounts. The fractions asialo- to trisialotransferrin were collected separately. The tetrasialo- to hexasialotransferrin were collected as a separate fraction. The asialo- and monosialo- fractions were not further diluted. The disialo- and trisialo- fractions were diluted 1:1 and the fraction containing the tetrasialo- to hexasialotransferrins was diluted 1:4 with mobile phase A.
To quantify the transferrin content of each isolated eluted fraction, the steps D1-D8 in the following immunoassay procedure were performed. In relation to the asialo fraction, in D8 the concentration was determined using calibrators from 0-2.5~.g/ml. In relation to the remaining fractions the steps D1-D8 were performed with the CDTect-calibrators.
Immunoassay procedure D1: 5001 calibrator is pipetted out. The calibrators are dilutions of human transferrin (obtained from WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 Intergen) in PBS containing 1% BSA, O.lo Tween 20 at pH 7.0) D2: 5001 sample is pipetted out D3 : 50.1 transferrin lzsl is added to the sample . The lzsl transferrin solution is made by dilution of lzsl labelled human transferrin (obtained from Isopharma AS, Kjeller, Norway) to a concentration suitable for gamma-counting in an aqueous buffered solution containing 0.037 M disodiumhydrogen phosphatedihydrate, 0.013 M sodiumdihydrogen phosphatemonohydrate, to BSA, 0.1% Tween 20 and 0.03% Patent Blue.
D4: 501 antibody is added. The antibody solution is made from rabbit anti human transferrin antiserum (obtained from BioCell, product No. 01090) diluted 1:450 in 0.4 M sodium phosphate buffer with to BSA, O.lo Tween 20 at pH 7Ø
D5: 2000.1 decanting suspension is added. The decanting suspension is a solution of secondary anti rabbit antibodies (obtained from Pharmacies &
Upjohn, product No. 30-3794-00) D6: incubate for 1 hour D7: centrifuge 10 mins at 1500xg and remove supernatant D8: determine radioactivity The concentration and o distribution of transferrin variants determined in a given serum sample is shown in Table 1 below (1501 serum is diluted to 1.1 ml and 800,1 is injected on the HPLC; dilution factor:
1.1/0.15*1/0.8 = 9.17):
Table 1 asialo mono- di- tri- tetra higher fiPLC fraction volume4 2.5 5 5.5 27.5 (ml) Sample volume (~l) 500 500 250 250 125 Conc. in RIA sample 0.29 0.29 0.87 1.20 2.85 (/a.l/mg) Conc. corrected for 0.29 0.29 1.73 2.41 11.41 sample volume (~1/mg) Conc. per ml 1.14 0.73 8.67 13.24 313.66 fraction (~g/ml) Conc. in serum 10.47 6.66 79.50 121.45 2876.26 (~1/mg) % CDT-RIA 0.34 0.22 2.57 3.92 92.95 Example 2 Correlation between asialo- and disialotransferrins and lack of monosialotransferrin Using the HPLC/RIA method described in Example l, monsialotransferrin levels were found to be very low.
In addition, asialo- and disialotransferrin levels were found to correlate as follows:
D = A.a + b (wherein D represents disialotransferrin content of a serum sample;
A represents asialotransferrin content of a serum WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 sample;
a = 22.2; and b = 36 mg/1) This represents a squared correlation coefficient of 0.9153 (see attached Figure 1).
Using relative units (% total transferrin coefficients), the following values for a and b were determined:
a = 22.25 ' b = 1.05% (0.0105) This represents a squared correlation coefficient of 0.9336 (see attached Figure 2).
Example 3 Determination of algorithm to calculate levels of asialotransferrin, disialotransferrin and CDT in a serum sample At least two solutions having known levels of asialo-and disialotransferrins are subjected to a separation method capable of removing substantially all tri- and higher sialotransferrins (e.g. based on lectins or ion exchange matrices). Thereafter the total transferrin content of the isolated fraction of transferrin is determined (e.g. by a radioimmunoassay method for transferrin quantitation). Based on the previously determined correlation between the level of asialo- and disialotransferrins and the low level of monoasialotransferrin, the following relations can be established:
T1 = c.Al + d.Dl T2 - c.A2 + d.D2 WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 T3 - c.A3 + d.D3 etc.
(wherein T1, T2, T3, etc. represent the measured total transferrin content of each sample following separation*;
A1, A2, A3, etc. represent the known contents of asialotransferrin in each sample;
D1, D2, D3, et,c. represent the known contents of diasialotransferrin in each sample; and c and d are constants).
* these may be measured in mass units or relative to the total transferrin content of the sample.
Calculation of the content of asialotransferrin, diasialotransferrin and CDT in any sample from the partial measurement of T:
For any sample tested:
T = c.A + d.D
and D = a.A + b (wherein T represents measured total transferrin content;
A represents actual asialotransferrin content in the sample;
D represents actual disialotransferrin content in the sample; and a, b, c and d are each constants).
It follows that:
WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 A = (T-d.b) / (c + d. a) ;
D = b + a(T-d.b)/(c + d.a); and CDT = A + D = b + (a + 1) (T-d.b) / (c + d.a) (where CDT is the total content of asialo-, monosialo-and disialotransferrin in the sample).
Example 4 Quantitation of transferrin content by means of anion exchange and lectin binding 20 ~.1 of a serum sample is mixed with 50 ~.l of a solution of 10 mM bis-tris, 3.1 mM sodium azide, 0.05%
Tween 20, 1 M HCl ~d pH 7.0, 0.8 mM Tris base, 0.15 mM
FeCl3, 0.15 M sodium citrate and 0.4 mM malefic acid.
The resulting solution is added to 0.5 ml of 250 preswollen Whatman QA52 ion exchange resin suspended in 20 mM Bis-Tris buffer ((2-hydroxy)amino-tris(hydroxymethyl)methane) pH = 6.3. The chloride content of the medium is carefully adjusted to retain substantially all transferrin molecules with more than two sialic acid residues (this may be monitored by HPLC
or isoelectric focusing). Thereafter, 0.25 ml of a 25%
suspension elderberry bark lectin (Vector Laboratories, US) is added (this ensures a more complete mopping up of sialylated transferrin molecules), and the suspension is mixed gently. The suspension is thereafter filtered by centrifugation in a Millipore Ultra-Free UFC3 OHV filter cup, and the filtrate is collected. 200 ~.1 of the filtrate is mixed with 200 ~,l of a transferrin antibody (Dako) solution diluted 1:10 in 0.27 M Tris, 4.5% PEG
8000, 4.3 mM sodium azide pH = 7.4. The concentration of transferrin in the filtrate is determined by interpolating the nephelometric signal in a standard curve constructed from standards of known concentrations of human transferrin.
With this separation method, c = 0.93 and d = 0.45.
Example 5 Quantitation of transferrin content by means of anion exchange 20 ~.l of a serum sample is mixed with 50 ~.l of a solution of 10'mM bis-tris, 3.1 mM sodium azide, 0.050 Tween 20, 1 M HCl ad pH 7.0, 0.8 mM Tris base, 0.15 mM
FeCl3, 0.15 M sodium citrate and 0.4 mM malefic acid.
The resulting solution is added to 0.5 ml of 250 preswollen Whatman QA52 ion exchange resin suspended in mM Bis-Tris buffer ((2-hydroxy)amino-tris(hydroxymethyl)methane) pH = 6.3. The chloride content of the medium is carefully adjusted to retain substantially all transferrin molecules with more than 20 two sialic acid residues (this may be monitored by HPLC
or isoelectric focusing). The suspension is thereafter filtered by centrifugation in a Millipore Ultra-Free UFC3 OHV filter cup, and the filtrate is collected. 200 ~.1 of the filtrate is mixed with 200 ~.l of a transferrin antibody (Dako) solution diluted 1:10 in 0.27 M Tris, 4.5o PEG 8000, 4.3 mM sodium azide pH = 7.4. The concentration of transferrin in the filtrate is determined by interpolating the nephelometric signal in a standard curve constructed from standards of known concentrations of human transferrin.
Example 6 Quantitation of transferrin content by means of immobilised lectiwfrom Sambuccus nigra in a column format.
a. 10 ~,l serum samples are mixed with 0.5 ml binding buffer 20 mM Tris-HC1 buffer with pH = 7.5 containing 150 mM sodium chloride.
b. Each diluted serum sample is passed through a column of~0.5 ml agarose elderberry bark (Sambuccus nigra) le~ctin (supplied by Vector Laboratories, Burlingame, USA) suspended in 20 mM Tris-HCl buffer with pH = 7.5 containing 150 mM sodium chloride, and another 1.0 ml of the same buffer is passed through the column.
c. Mix 200 ~.1 of the eluted solution with 200 ~,1 of an anti-transferrin antibody solution comprising 0.27 M Tris, 4.5% PEG 8000, 4.3 mM sodium azide, 1:10 dilution of Dako anti-human-transferrin antibodies Q0327, and HC1 to pH = 7.4.
d. Read the turbidimetric/nephelometric signal.
With this separation method, c = 0.98 and d = 0.08.
Exam 1p a 7 Equipment - Columns (7mm internal diameter) containing 0.5m1 POROS HQ50 (Perseptive Biosystems) between polyethylene upper and lower porous frits (Porex, Atlanta, Ga, USA) - columns available from Pierce Company, USA
- Four calibrators**
- Pipettes covering volumes from 4~,1 to 3m1 Alternatively: multipipettes for volumes 1001, 200,1, 2m1 and 3m1 WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 - Racks (for tubes (75 x l2mm) - Microtiter plates -- Reader for microtiter plates, 405nm filter ** Prepared using human normal serum. See Table 2 for dilutions Table 2 Calibrators (mg/ml) Human normal serum Solution 2 containing 2.17 mg transferrin/ml C1:0.002 100.1 C3 900.01 C2:0.01 4.6.1 995.4,1 C3 :0.02 10.1.1 1089.9~C1 C4:0.03 13.81 986.2.1 (Calibrator C1 is produced by dilution of calibrator C3 with Solution 2).
Reagents - Solution 1 lOmM BisTris (bis(2-hydroxyethyl)amino-tris-(hydroxymethyl)methane) 3.lmM Sodium Azide 0.05% Tween 20 1M HC1 ad pH 7.0 0.8mM Tris base (Tris(hydroxymethyl)aminomethane) 0.15mM FeCl3 0.15mM Sodium Citrate 0.4mM Malefic acid deionized HZO a.s.
Solution 2 50mM BisTris (bis(2-hydroxyethyl)amino-tris-(hydroxymethyl)methane) WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 3.lmM Sodium Azide 0.05% Tween 20 1M HCl ad pH 6.00 approx 3 mM NaCl added deionized H20 ,~.s.
Turbidimetric reaaent 900.1 0.3M Tris/PEG pH 7.4 100.1 Rabbit anti-serum transferrin (Dako) The 0.3M Tris/PEG pH 7.4 comprises:
0.3m Tris. HC1 6% Polyethylene glycol (PEG 8000) 3.lmM Sodium Azide 2M NaOH ~d pH 7.4 deionized HZO a.s.
Preparation of columns:
Use one column for each sample to be tested. Elute surplus transport buffer by removing first the top and thereafter the bottom stopper, discard and eluate.
Prepared columns should be used within 2 hours.
Sample testina procedure - Sample procedure 1. Add 100,1 serum to 500.1 solution 1 in a test tube. Mix.
2. Incubate for 5-10 minutes at ambient temperature.
- Column separation 1. All solutions added must elute freely from the column.
2. Add 500.1 incubated sample to a column.
3. Let the sample sink into the top filter before adding l.Oml of solution 2.
4. Let solution 2 sink into the top filter.
Change tubes below columns. Solution eluted up to this point should be discarded. Add 2.0m1 of solution 2 to each column. Collect 2m1 eluate 2.
- Measurement 1. Add 200,1 of each calibrator, and 200,1 of eluate 2 from the column separation to separate wells of a microtiter plate. Read at 405nm.
2. Add 100.1 turbidimetric reagent to each well.
3. Incubate for 15 minutes at ambient temperature.
4. Read Y~esults using a reader with 405nm filter and subtract the background calculated in step 1.
5. Establish a calibration curve using non-linear regression.
6. transferrin content in the serum sample is calculated from the calibration curve.
With this separation method, c = 0.98 and d = 0.66.
Example 8 Determination of asialo- and disialo- transferrin, and a combination of asialo-, monosialo- and disialo-transferrin, using a combined HPLC-RIA method.
A further experiment was carried out to determine the correlation between 1) asialo- and monosialo- transferrin isoforms in serum samples, 2) asialb- and disialo- transferrin isoforms in serum samples, 3) (asialo-, monosialo- and disialo-) and asialo-transferrin isoforms in serum samples, and 4) (asialo-, monosialo- and disialo-) and disialo-transferrin isoforms in serum samples.
These experiments were carred out to test the reliability of the relationship between the content of asialo-, mono sialo- and disialo- transferrins (in the above combinations 1-4) in serum samples from a number of different individuals.
REAGENTS, MATERIALS, INSTRUMENTS AND EQUIPMENT
The experiment was carried out using the same reagents, materials, instruments and equipment as described in Example 1.
EXPERIMENTAL
Transferrin in lipid stripped, iron treated serum was analysed on two combined methods, HPLC and RIA. The different sialic acid isoforms were determined, first by separation on a HPLC with a ion exchange column. The o of the different fractions are first determined on the HPLC, then the concentration of the fractions are determined on a gamma counter by using parts of the CDTectTM
RESULTS AND DISCUSSION
Fourteen samples of serum were collected from 14 individuals and in each of these samples, the asialo-, mono- and disialo - transferrin fractions were separated and collected individually. For each sample, the concentration of each fraction was determined as percentage of total transferrin using the RIA method (%CDT-RIA) and HPLC method (%CDT-HPLC) as described in Example 1. The concentration of the asialo-, mono- and di-sialo transferrin was also determined in ~.g/mL for the RIA method'(CDT RIA ~.g/ML). These results are shown below in Table 3.
WO 01/42795 CA 02393556 2002-06-06 pCT/GB00/04732 CDT-RIA ~ CDT
CDT-HPLC RIA
~g/m sialomonodi tri etra+sialomonodi ri etrapentasialomonodi SB 0.18 0.3 1.695 92.820 0.151.285 75.4818.1 0.4 0.525.4 CDT 0.34 0.49.6 7.0487.520.180.15.82 5.3972.2419.22.68 0.779.2 4 0. 0.43. 8 88 0 0 1. 7 76. 14 . 0. .
18 08 .46 . .24 32 . 59 . 36 65 04 123 0.33 0.39.11 6.7788.410.190.14.86 6.9859.420.42 .84 0.9910.6 2 1.07 0.7 10.68.0779.590.930.389 8.8 58.921.98 .92 1.2418.9 122 0.43 0 . 6.4288 0. 0 3 5 67 3 .26 0. 13 . 61 . 35 . . .24 . . 64 .4 7 0. 0. 1 9. 87. 0. 0 1 8 68 1 .41 1 5 13 39 . 93 74 03 . . .41 .23 .21 .23 .
126 0.51 0.345.52.46 89.170.530.155.323.9263.326.76 .34 1.2219.7 127 0 0 1 . 93 0 0 1 .49 68 4 . 0 7 . . . 66 . . . . . . 83 . .
BL 0 0 1 5 92 0 0 0 . 76. 18 0 0. 3 .41 . . . . . 12 79 . 85 .
129 0.41 0.45.41 .37 90.370.350.19.59 3.9967.423.47 .23 1.3413.2 130 0.88 0.718.097.2 83.130.910.247.326.9470.0114.58.55 1.2514.2 136 0.13 0.191.3 1.3497.040 0.131.04.63 2.961.25 .44 0.664.6 125 0.35 0.49.87 7.1687.130.110.273.237.4868.80.12 .94 1.3 12.9 Figure 3 shows separation of transferrin isoforms by HPLC. The separation of monosialo and disialo is not complete, as can be seen between 9 and 10 min.
Figures 4A and 4B show correlation of asialo vs monosialo, in A determined in o transferrin, in B
determined in ~,g/mL. The squared correlation coefficient based on percentage determinations (Fig. 4A) was 0.5235. Based on mass units (Fig. 4A) the squared correlation coefficient was 0.3339. The graphs are produced using data from the RIA method.
Hence the asialo vs monosialo transferrin did.not show a good correlation for either of the determinations. One of the reasons for this might be that for the higher asialo values the disialo peak seems SUBSTITUTE SHEET (RULE 26) to overlap the monosialo. This can be seen in Figure 3.
Figures 5A and 5B show correlation of asialo vs disialo, in A determined in o transferrin, in B
determined in ~.g/mL. The graphs are produced using data from teh RIA method.
The squared correlation coefficient based on percentage determination (relative units) was 0.9501 (see Fig. 5a). Based on mass units (Fig. 5B) the squared correlation coefficient was 0.8603.
The correlation of asialo- vs disialo- transferrin was as expected better for the % determination than for ~g/mL. This is due to the correction for high and low total transferrin for high and low total transferrin content, in the % isoform determination.
Figure 6 shows the correlation of (asialo-, monosialo- and disialo- transferrin) with asialo transferrin, determined in mass units (~,g/mL). The squared correlation coefficient was 0.8797. The graph is produced using data from the RIA method.
Figure 7 shows the correlation of (asialo-, monosialo- and disialo- transferrin) with disialotransferrin, determined in mass units (~g/mL).
The squared correlation co-efficient was 0.9176. The graph is produced using data from the RIA method.
At present, the precision of asialotransferrin determination is lower than e.g. disialotransferrin (due to much lower concentration), and therefore the correlation coefficient for asialotransferrin is lower.
However the correlation coefficient for asialotransferrin is much higher than had previously been thought possible.
Example 9 Determination of asialo, monosialo, disialo and trisialo, and combinations of asialo, monosialo and disialo transferrin, using a combined HPLC-RIA method.
A further experiment was carried out to determine the correlation between 1) asialo- and (monosialo- and disialo-) transferrin isoforms in serum samples, 2) asial,b- and trisialo- transferrin isoforms in serum samples, 3) (asialo-, monosialo- and disialo-) and asialo-transferrin isoforms in serum samples, and 4) (asialo-, monosialo- and disialo-) and disialo-transferrin isoforms in serum samples.
These experiments were carred out to test the reliability of the relationship between the content of asialo-, mono sialo- and disialo- transferrins (in the above combinations 1-4) in serum samples from a number of different individuals.
REAGENTS, MATERIALS, INSTRUMENTS AND EQUIPMENT
The experiment was carried out using the same reagents, materials, instruments and equipment as described in Example 1.
EXPERIMENTAL
Transferrin in lipid stripped, iron treated serum was analysed on two combined methods, HPLC and RIA. The different sialic acid isoforms were determined, first by separation on a HPLC with a ion exchange column. The o of the different fractions are first determined on the HPLC, then the concentration of the fractions are determined on a gamma counter by using parts of the CDTectTM.
RESULTS AND DISCUSSION
Twenty six samples of serum were taken from 26 individuals. However the mono- and disialo-transferrin fractions were collected together as a single fraction.
The concentrations of each fraction were determined by the RIA and HPLC methods respectively and the results are shown in Table 4 below:
% CDT-RIA % CDT
HPLC
~g/mL
sialo+ tri etra entasialo + tri etra penta di di ampl a 1.5 2.1 5.5 63.2 17.81.3 11.74.6 3.4 19 ample 1.2 0.5 4.2 67.8 15.21.4 11.34.7 4.6 17.1 ample 0.5 3.7 6.3 73.5 16 0.6 3.8 6.1 2.8 16.7 ample 0 ~2 4.6 75.4 18 0.6 2.6 4.7 0.2 22 10 ample 0 2.6 3.1 70 4.4 0.4 3 3.1 4.7 8.8 ample 0.5 4.1 5.1 69.4 0.8 0.4 3.9 4.8 7.1 3.7 ample 0 1.6 6.12 76 16.30.5 1.3 5.7 7.6 14.9 ample 0 2.8 5.5 73.6 18.10.4 2.7 4.7 4.3 17.8 ample 0 2.9 5.4 73.3 18.40.2 3.1 4.9 2.4 19.4 1 ample 0.3 3.8 6 73.6 16.30.7 3.8 5.3 1.8 18.4 ample 0 1.5 2.2 74.1 2.2 0.5 1.5 2.2 5.6 0.3 ample 0.4 3.1 5.9 72 18.50.8 3.4 5.6 1.3 18.9 Figure 8 shows the correlation of asialo 20 transferrin with (monosialo- and disialo-) transferrin, determined in o transferrin. A reasonable correlation was found. The squared correlation coefficient was 0.8596. The graphs are produced using data from the RIA
method.
Figure 9 shows the correlation of asialo- with trisialo- transferrin, determined in % transferrin. As expected, no correlation was found. The squared correlation coefficient was very low, at 0.01754. The graph is produced using data from the RIA method.
Figure 10 shows the correlation of (asialo-, monosialo- and disialo-transferrin) with asialotransferrin, determined in o transferrin. The squared correlation coefficient was 0.9278. The graph is produced using data from the RIA method.
Figure 11 shows the correlation of (asialo-, monosialo- and disialo- transferrin) with disialotransferrin, determined in % transferrin. The squared correlation coefficient was 0.9906. The graph is produced using data from the RIA method.
CONCLUSION
From these Examples 8 and 9 it can be seen that there is a correlation between asialo and mono- & di-sialotransferrin, as well as between asialo- and disialo- transferrin and between either asialo- or disialo- transferrin and CDT (asialo- + monosialo- +
disialo- transferrin). Important to notice is that the monosialo fraction is small compared to disialo and will therefore only give a small contribution in the correlation study. This indicates that both asialo and disialo isoforms of transferrin can be used as parameters for monitoring alcoholic diseases. Trisialo was not seen to give any correlation to asialo and is probably not a good marker. Hence the clinical value of the assay according to the invention has been demonstrated, and combinations of transferrin variants suitable for use in producing the algorithm according to the invention have been found.
Furthermore, Example 9 illustrates that complete separation of the disialo transferrin is not required because the presence of the monosialo-variant in the determined or separated fraction is not harmful to the correlation results.
Claims (19)
1. A method of producing an algorithm for determining the content of a transferrin variant or combination of transferrin variants, preferably a CDT variant or combination of CDT variants, in a sample of body fluid, said method comprising:
(a) obtaining at least two solutions each having known contents of asialo- (A1, A2, A3, etc.) and disialo-transferrins (D1, D2, D3, etc.);
(b) determining the content in each of said solutions of a transferrin variant or combination of transferrin variants fractions substantially free from tri- and higher sialylated transferrins;
(c) determining the total transferrin variant content (T1, T2, T3, etc.) of said fractions; and (d) producing an algorithm capable of determining the content of any transferrin variant or combination of transferrin variants, preferably a CDT variant or combination of CDT variants, in any given sample of body fluid subjected to said determination step b) .
(a) obtaining at least two solutions each having known contents of asialo- (A1, A2, A3, etc.) and disialo-transferrins (D1, D2, D3, etc.);
(b) determining the content in each of said solutions of a transferrin variant or combination of transferrin variants fractions substantially free from tri- and higher sialylated transferrins;
(c) determining the total transferrin variant content (T1, T2, T3, etc.) of said fractions; and (d) producing an algorithm capable of determining the content of any transferrin variant or combination of transferrin variants, preferably a CDT variant or combination of CDT variants, in any given sample of body fluid subjected to said determination step b) .
2. A method as claimed in claim 1 wherein said determination step b) comprises subjecting each of said solutions to a separation method whereby to separate fractions substantially free from tri- and higher sialylated transferrins.
3. A method as claimed in claim 1 or 2 wherein said algorithm is capable of determining the actual content or amount of asialo- or disialo-transferrin.
4. A method as claimed in any one of claims 1 to 3 wherein said algorithm is capable of determining the actual content or amount of asialo-, monosialo- and disialo-transferrins.
5. A method as claimed in any one of claims 1 to 4 wherein said algorithm is based upon a quantitation of each of the isoforms of transferrin having zero, one or two sialic acid residues per molecule.
6. A method as claimed in any one of claims 1 to 5 wherein said algorithm is produced on the basis of the high correlation between the content of asialo- and disialo-transferrins in a sample, preferably also the low level of monosialotransferrin.
7. A method as claimed in any one of claims 1 to 6 wherein said algorithm is defined by at least one of the following equations:
A = (T-d.b) / (c + d.a) D = b + a(T-d.b)/(c + d.a) CDT = A + D = b + (a + 1) (T-d.b) / (c + d.a) (wherein T represents the determined total transferrin content in teh determined fraction;
A represents the actual asialotransferrin content in the sample;
D represents the actual disialotransferrin content in the sample;
CDT represents the actual total content of asialo-, monosialo- and disialotransferrins in the sample;
a and b are constants defining the correlation between A
and D in any serum sample; and c and d are constants specific to the determination step b) .
A = (T-d.b) / (c + d.a) D = b + a(T-d.b)/(c + d.a) CDT = A + D = b + (a + 1) (T-d.b) / (c + d.a) (wherein T represents the determined total transferrin content in teh determined fraction;
A represents the actual asialotransferrin content in the sample;
D represents the actual disialotransferrin content in the sample;
CDT represents the actual total content of asialo-, monosialo- and disialotransferrins in the sample;
a and b are constants defining the correlation between A
and D in any serum sample; and c and d are constants specific to the determination step b) .
8. A method for determining the content of a transferrin variant or combination of transferrin variants, preferably a CDT variant or combination of CDT
variants, in a body fluid for use in the assessment of alcohol consumption, said method comprising:
(a) determining the content in a sample of said body fluid of a transferrin variant or combination of transferrin variants a fraction substantially free from tri- and higher sialylated transferrins;
(b) determining the total transferrin variant content in said fraction; and (c) determining the content of any transferrin variant or combination of transferrin variants, preferably the content of any CDT variant or combination of CDT variants, in said sample using an algorithm obtained in accordance with a method as herein described.
variants, in a body fluid for use in the assessment of alcohol consumption, said method comprising:
(a) determining the content in a sample of said body fluid of a transferrin variant or combination of transferrin variants a fraction substantially free from tri- and higher sialylated transferrins;
(b) determining the total transferrin variant content in said fraction; and (c) determining the content of any transferrin variant or combination of transferrin variants, preferably the content of any CDT variant or combination of CDT variants, in said sample using an algorithm obtained in accordance with a method as herein described.
9. A method as claimed in claim 8, wherein said determination step a) comprises subjecting a sample of said body fluid to a separation method capable of separating a fraction substantially free from tri- and higher sialylated transferrins.
10. A method as claimed in any one of claims 1 to 9 wherein said body fluid is blood or a blood-derived sample.
11. A method as claimed in any one of claims 1 to 10 wherein, the fraction substantially free from tri- and higher sialylated transferrins comprises at least 60% of the asialo- and monosialo- transferrin variants present in the sample prior to separation.
12. A method as claimed in any one of claims 1 to 10 wherein the fraction substantially free from tri- and higher sialylated transferrins has a disialotransferrin content of at least 20%, preferably up to 60 to 70% of the total disialotransferrin content of the sample prior to separation.
13. A method as claimed in any one of claims 1 to 10 wherein the fraction substantially free from tri- and higher sialylated transferrins comprises less than 20%
asialotransferrin, less than 5% monosialotransferrin and 70 to 80% disialotransferrin.
asialotransferrin, less than 5% monosialotransferrin and 70 to 80% disialotransferrin.
14. A method as claimed in any one of claims 1 to 13 wherein at least 70 to 80% of the transferrin molecules in the fraction substantially free from tri- and higher sialylated transferrins carry a carbohydrate chain or a residue thereof.
15. A method as claimed in any one of claims 2 to 7 and 9 to 14 wherein said separation method comprises the steps of contacting a sample of body fluid with a carbohydrate-binding ligand, preferably a lectin or mixture of lectins, followed by separation of a fraction not binding to said ligand.
16. A method as claimed in claim 15 wherein said carbohydrate-binding ligand is a sialic acid binding lectin
17. A kit for a diagnostic assay for the assessment of alcohol consumption, said kit comprising:
means for subjecting a sample of body fluid to a determination step capable of determining the content of target transferrin variants in a fraction substantially free from tri- and higher sialylated transferrins;
means for the detection of transferrin; and means for determining the content of any transferrin variant or combination of transferrin variants in a sample of body fluid subjected to said determination step.
means for subjecting a sample of body fluid to a determination step capable of determining the content of target transferrin variants in a fraction substantially free from tri- and higher sialylated transferrins;
means for the detection of transferrin; and means for determining the content of any transferrin variant or combination of transferrin variants in a sample of body fluid subjected to said determination step.
18. A kit as claimed in claim 17 wherein said kit further comprises at least two transferrin solutions having known asialo- and disialo- concentrations.
19. A kit as claimed in claim 17 wherein said means for determining the content of a fraction substantially free from tri- and higher sialylated transferrins comprises means for subjecting a sample of body fluid to a separation method capable of producing a fraction substantially free from tri- and higher sialylated transferrins.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9929308.6 | 1999-12-10 | ||
| GBGB9929308.6A GB9929308D0 (en) | 1999-12-10 | 1999-12-10 | Assay |
| PCT/GB2000/004732 WO2001042795A2 (en) | 1999-12-10 | 2000-12-11 | Transferrin assay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2393556A1 true CA2393556A1 (en) | 2001-06-14 |
Family
ID=10866134
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002393556A Abandoned CA2393556A1 (en) | 1999-12-10 | 2000-12-11 | Transferrin assay |
Country Status (11)
| Country | Link |
|---|---|
| US (1) | US7166473B2 (en) |
| EP (1) | EP1247101B1 (en) |
| JP (1) | JP2003516543A (en) |
| AT (1) | ATE283488T1 (en) |
| AU (1) | AU1871001A (en) |
| CA (1) | CA2393556A1 (en) |
| DE (1) | DE60016281T2 (en) |
| DK (1) | DK1247101T3 (en) |
| ES (1) | ES2233484T3 (en) |
| GB (1) | GB9929308D0 (en) |
| WO (1) | WO2001042795A2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2002307776A1 (en) * | 2002-04-16 | 2003-10-27 | Kamada Ltd. | Ultrapure transferrin for pharmaceutical compositions |
| US7691640B2 (en) * | 2005-09-30 | 2010-04-06 | Adeline Vanderver | Biochemical marker for diagnosing a leukodystrophy |
| EP2440928A4 (en) * | 2009-06-08 | 2012-12-12 | Acrotech Systems Inc | Rapid detection of cerebrospinal fluid, methods and systems therefore |
| WO2011052804A1 (en) * | 2009-10-30 | 2011-05-05 | Tokyo University Of Science Educational Foundation Administrative Organization | Method of delivering agent into target cell |
| RU2461832C2 (en) * | 2010-07-02 | 2012-09-20 | Государственное образовательное учреждение Высшего профессионального образования "Омская государственная медицинская академия Федерального агентства по здравоохранению и социальному развитию Росздрава" | Diagnostic technique for alcohol abuse |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE440699B (en) * | 1984-02-06 | 1985-08-12 | Pharmacia Ab | ASSUME MEDIUM ISOTRANSFERRIN DETERMINATION ESTIMATE AN INDIVIDUAL ALCOHOL CONSUMPTION |
| DK0535162T3 (en) * | 1990-06-21 | 1995-06-12 | Axis Biochemicals As | Analysis for analyte variants |
| SE501668C2 (en) * | 1993-08-06 | 1995-04-10 | Biolin Medical Ab | Quantification of CD transferrin at high alcohol consumption by HPLC |
| DK0811166T3 (en) * | 1995-02-22 | 2000-03-27 | Axis Biochemicals Asa | Carbohydrate-poor transferrin analysis |
| US5798212A (en) * | 1995-02-22 | 1998-08-25 | Axis Biochemicals Asa | CDT assay |
| GB9713559D0 (en) * | 1997-06-26 | 1997-09-03 | Axis Biochemicals Asa | Assay |
| GB9827411D0 (en) * | 1998-12-11 | 1999-02-03 | Axis Biochemicals Asa | Dipstick assay |
-
1999
- 1999-12-10 GB GBGB9929308.6A patent/GB9929308D0/en not_active Ceased
-
2000
- 2000-12-11 AU AU18710/01A patent/AU1871001A/en not_active Abandoned
- 2000-12-11 WO PCT/GB2000/004732 patent/WO2001042795A2/en active IP Right Grant
- 2000-12-11 AT AT00981475T patent/ATE283488T1/en not_active IP Right Cessation
- 2000-12-11 DK DK00981475T patent/DK1247101T3/en active
- 2000-12-11 DE DE60016281T patent/DE60016281T2/en not_active Expired - Fee Related
- 2000-12-11 ES ES00981475T patent/ES2233484T3/en not_active Expired - Lifetime
- 2000-12-11 CA CA002393556A patent/CA2393556A1/en not_active Abandoned
- 2000-12-11 EP EP00981475A patent/EP1247101B1/en not_active Expired - Lifetime
- 2000-12-11 JP JP2001544033A patent/JP2003516543A/en not_active Withdrawn
-
2002
- 2002-06-10 US US10/167,983 patent/US7166473B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| DE60016281D1 (en) | 2004-12-30 |
| US20030087450A1 (en) | 2003-05-08 |
| EP1247101B1 (en) | 2004-11-24 |
| WO2001042795A2 (en) | 2001-06-14 |
| ATE283488T1 (en) | 2004-12-15 |
| US7166473B2 (en) | 2007-01-23 |
| JP2003516543A (en) | 2003-05-13 |
| ES2233484T3 (en) | 2005-06-16 |
| GB9929308D0 (en) | 2000-02-02 |
| DE60016281T2 (en) | 2005-12-22 |
| AU1871001A (en) | 2001-06-18 |
| WO2001042795A3 (en) | 2002-05-10 |
| DK1247101T3 (en) | 2005-03-29 |
| EP1247101A2 (en) | 2002-10-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6716641B1 (en) | Dipstick for carbohydrate-free transferrin assay | |
| Salmela et al. | Carbohydrate‐deficient transferrin during 3 weeks' heavy alcohol consumption | |
| AU654497B2 (en) | Analyte variant analysis | |
| US20090117668A1 (en) | Immune Agglutination Reagent Kit and Method of Measuring Antigen | |
| US4624916A (en) | Process and composition for the rapid quantitation of small levels of creative kinase-MB isoenzyme | |
| US20050239137A1 (en) | Assay for carbohydrate-free transferrin | |
| EP0968429B1 (en) | Method and kit for determining the phenotype of a haptoglobin and use thereof | |
| EP1247101B1 (en) | Transferrin assay | |
| US6103478A (en) | CDT assay | |
| US5798212A (en) | CDT assay | |
| JP4753366B2 (en) | % CDT quantification method | |
| JP2003277398A (en) | Antibody against transferrin-including immune complex, method of producing the same, hybridoma and immunity measuring method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |