WO1983000877A1 - Glucose oxidase immunohistochemical detection of antinuclear antibodies - Google Patents
Glucose oxidase immunohistochemical detection of antinuclear antibodies Download PDFInfo
- Publication number
- WO1983000877A1 WO1983000877A1 PCT/US1982/001107 US8201107W WO8300877A1 WO 1983000877 A1 WO1983000877 A1 WO 1983000877A1 US 8201107 W US8201107 W US 8201107W WO 8300877 A1 WO8300877 A1 WO 8300877A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antigen source
- contacting
- source
- sheath
- support
- Prior art date
Links
- 235000019420 glucose oxidase Nutrition 0.000 title claims abstract description 10
- 108010015776 Glucose oxidase Proteins 0.000 title claims abstract description 9
- 239000004366 Glucose oxidase Substances 0.000 title claims abstract description 9
- 229940116332 glucose oxidase Drugs 0.000 title claims abstract description 9
- 230000003460 anti-nuclear Effects 0.000 title claims description 15
- 238000013115 immunohistochemical detection Methods 0.000 title abstract description 3
- 238000000034 method Methods 0.000 claims abstract description 32
- 239000000427 antigen Substances 0.000 claims abstract description 20
- 102000036639 antigens Human genes 0.000 claims abstract description 20
- 108091007433 antigens Proteins 0.000 claims abstract description 20
- 238000012360 testing method Methods 0.000 claims abstract description 17
- 239000000203 mixture Substances 0.000 claims abstract description 11
- 210000002966 serum Anatomy 0.000 claims abstract description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 4
- 239000008103 glucose Substances 0.000 claims abstract description 4
- 102000019040 Nuclear Antigens Human genes 0.000 claims description 12
- 108010051791 Nuclear Antigens Proteins 0.000 claims description 12
- 239000000758 substrate Substances 0.000 claims description 11
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical group CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 108060003951 Immunoglobulin Proteins 0.000 claims description 7
- 102000018358 immunoglobulin Human genes 0.000 claims description 7
- ZYDGCYWJDWIJCS-UHFFFAOYSA-N 1-methoxyphenazine Chemical compound C1=CC=C2N=C3C(OC)=CC=CC3=NC2=C1 ZYDGCYWJDWIJCS-UHFFFAOYSA-N 0.000 claims description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 6
- 241000283707 Capra Species 0.000 claims description 5
- 239000003960 organic solvent Substances 0.000 claims description 5
- 229910001873 dinitrogen Inorganic materials 0.000 claims description 4
- 239000007789 gas Substances 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 210000005084 renal tissue Anatomy 0.000 claims description 4
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 claims description 3
- 230000001681 protective effect Effects 0.000 claims description 3
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 claims description 3
- 238000010186 staining Methods 0.000 claims description 3
- 229940072221 immunoglobulins Drugs 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 229920006267 polyester film Polymers 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 230000000087 stabilizing effect Effects 0.000 claims description 2
- 230000000984 immunochemical effect Effects 0.000 claims 1
- 125000003831 tetrazolyl group Chemical group 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 5
- 230000018044 dehydration Effects 0.000 abstract description 4
- 238000006297 dehydration reaction Methods 0.000 abstract description 4
- 239000003791 organic solvent mixture Substances 0.000 abstract description 2
- 208000005156 Dehydration Diseases 0.000 abstract 1
- 239000004698 Polyethylene Substances 0.000 abstract 1
- NIQQIJXGUZVEBB-UHFFFAOYSA-N methanol;propan-2-one Chemical compound OC.CC(C)=O NIQQIJXGUZVEBB-UHFFFAOYSA-N 0.000 abstract 1
- 229910052757 nitrogen Inorganic materials 0.000 abstract 1
- -1 polyethylene Polymers 0.000 abstract 1
- 229920000573 polyethylene Polymers 0.000 abstract 1
- 238000003556 assay Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 5
- 239000000463 material Substances 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 239000002250 absorbent Substances 0.000 description 3
- 230000002745 absorbent Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000000711 cancerogenic effect Effects 0.000 description 2
- 231100000315 carcinogenic Toxicity 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 239000000700 radioactive tracer Substances 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229920002799 BoPET Polymers 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 239000005041 Mylar™ Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000012120 mounting media Substances 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 210000001519 tissue Anatomy 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
- G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/552—Glass or silica
Definitions
- This invention relates generally to immuno ⁇ enzymatic processes for use in medical diagnoses, and more particularly, to an immunohistochemical process for the detection of antinuclear antibodies in test serum.
- an increasing amount of research has focused on improving clinical laboratory tests.
- a primary thrust of this research has been to replace the traditional radiotracer labels with safer and more stable labels.
- fluorescent markers have found wide acceptance. While fluorescent markers give quick results, they have not proven entirely satis- factory for a number of assays because once placed in a detectable state they have a very limited shelf life, fading after a few days or more. Moreover, fluorescent assays frequently exhibit high background due to auto-
- horseradish peroxidase has not proven entirely satis ⁇ factory, however, because of a high tendency to produce non-specific background stain, even in spite of attempts to quench the presence of endogenous peroxidase-like activity.
- Another objectionable feature of the peroxi ⁇ dase label is that it generally requires the use of a highly carcinogenic material, diaminobenzidine, for superior stain development. Although substitutes for this material have been developed, their contrast and intensity are typically not entirely satisfactory.
- the present invention provides an immunohisto- chemical method which substantially reduces the amount of background stain caused by endogenous materials, while producing a relatively permanent record of the assay. Moreover, the reagents of the present invention are relatively inexpensive to manufacture, are stable for long periods of time even when stored at room tempera ⁇ ture, and have minimal carcinogenic characteristics.
- an immuno ⁇ enzymatic process for the detection of a component of a test sample comprising steps of: contacting the test sample with an antigen source fixed onto support and stabilized by dehydration; contacting the antigen source with an antibody conjugated with glucose oxidase and reactive with the component; incubating the antigen source with the solution containing glucose and a chro ogenic mixture and; analyzing the antigen source for the presence of color from the incubation.
- the chromogenic mixtures preferably contain t-nitroblue tetrazolium chloride and m-phenazine metho- sulfate, which permit analysis with a light microscope.
- the component of the test sample is an antinuclear antibody
- the antigen source fixed on a glass slide is preferably Hep-2 cells or rat kidney tissue, and the conjugated antibody is goat immunoglobulin reactive with human immunoglobulins.
- an immunohistoche ical method for detecting antinuclear antibodies in a test serum, substantially in the absence of background stain ⁇ ing comprises steps of: providing a nuclear antigen source fixed onto a slide and dehydrated with acetone at about 4 * C;
- u ⁇ *- U contacting the nuclear antigen source with the test serum; contacting the nuclear antigen source with goat immunoglobulin conjugated with glucose oxidase and reactive with the .antinuclear antibodies; incubating the nuclear antigen source with a solution containing beta-D-glucose, t-nitroblue tetra- zolium, and 1-methoxyphenazine methosulfate; and analyzing the nuclear antigen source under a light microscope for the presence of a formazan stain.
- Another aspect of the invention is a method of stabilizing an im unochemical substrate fixed on a solid support, the method comprising the steps of: providing a substrate fixed on the support; contacting the substrate with an organic solvent reagent to substantially dehydrate the substrate; placing the support into a protective sheath; backfilling the sheath with a gas; and sealing the support in the sheath.
- the support is preferably a glass slide, and the substrate Hep-2 cells or rat kidney tissue.
- the organic solvent reagent can be composed of acetone, methanol, or mixtures thereof, and the interacting step performed between about 4 and 8° C.
- the sheath can be composed of a polyester film, and can contain a des- sicant.
- the preferred gas is nitrogen gas.
- Exemplary starting materials useful in practicing the present invention are slides fixed with human epethelial cells (Hep-2) or with rat kidney tissue suitable for use in antinuclear antibody assays.
- Such slides can be purchased commercially from a number of sources, including Antibodies Incorporated, Davis, California, and Behring, La Jolla, California, or, alternatively, can be manufactured according to con- ventional techniques. Generally, these slides should be stored at -20°C. to prevent degradation.
- the slides are immersed ' for about 10 minutes in a bath containing histological grade acetone, methanol, or a combination of these organic solvents.
- the bath is maintained at a temperature preferably ranging from about 4 C. to about 8 C.
- After removing the slides from the bath they are allowed to air dry for about 2 to 3 minutes.
- the slides are preferably stored at about 5°C. during continued processing.
- the slides are placed into a protective sheath, preferably made from a poly ⁇ ethylene film, such as MYLAR bags with an aluminum outer layer.
- a protective sheath preferably made from a poly ⁇ ethylene film, such as MYLAR bags with an aluminum outer layer.
- a small packet containing a standard dessicant is placed in the bag, and the bag is partially sealed. Nitrogen gas is then introduced through the unsealed portion of the bag until the bag bulges slightly. The bag is then totally sealed to prevent the nitrogen gas from escaping.
- O PI Goat immunoglobulin conjugated with the glucose oxidase enzyme can be prepared as follows. Glucose oxidase, which is available from a variety of commercial sources, including Sigma, St. Louis, Missouri, and Boehringer Manneheim, Indianapolis, Indiana, is dissolved in distilled water at a concentration of about 12 mg/ml. To this solution is added about 0.2 ml of ,0.1 M sodium periodate. The mixture is stirred for about 20 minutes and then dialyzed, preferably against 1 mM sodium acetate buffer, pH 4.4, for about 16 hours.
- the glucose oxidase solution is mixed with 1 ml of 0.01 M sodium bicarbonate buffer, pH 9.5, containing about 8 mg of the IgG fraction of goat anti- human im unoglobulins (IgG, IgA, and IgM) .
- 0.1 mis of. an aqueous solution containing about 4 mg/ml sodium borohydrate water solu ⁇ tion is added.
- the entire mixture can be chromatographed on a gel filtration column, such as a Sephacryl S-300, available from Pharmacia, Piscataway, New Jersey, and the fractions containing the enzyme-IgG conjugate pooled. This pool can then be titrated. to determine working dilutions.
- a method for detecting antinuclear antibodies utilizing these reagents is performed as follows.
- a small amount of test serum, or other test solution, is contacted with the nuclear antigen fixed source on the slide. After about thirty minutes at room temperature, the serum is washed from the slide with phosphate buf ⁇ fered saline (PBS) , and then soaked in a cold PBS solu ⁇ tion for about 10 minutes. The slides are removed from the PBS and excess moisture blotted with absorbent paper.
- a small amount of the enzy e-IgG conjugate, preferably about 50 ul, is contacted with the antigen source for about 30 minutes at room temperature. The slide is again washed with PBS, soaked for 10 minutes in a cold PBS solution, removed, and excess moisture blotted with absorbent paper.
- the color reagent may be pre ⁇ pared. It consists of about 2 volumes beta-D-glucose, 15 mg/ml in 0.1 M sodium phosphate buffer, pH 6.9, one volume t-nitroblue tatrazolium (t-NBT) , 2 mg/ml in the same buffer (if necessary, this may be filtered through Whattman No. 1 filter paper just prior to use) , and 1 volume of 1-methoxyphenazine methosulfate (m-PMS), 0.8 mg/ml in the same buffer.
- t-NBT t-nitroblue tatrazolium
- m-PMS 1-methoxyphenazine methosulfate
- the preferred chro o- genic reagents are t-NBT and m-PMS, other materials can also be utilized.
- the mixture may be prepared about 1 hour before the staining reaction.
- a small volume, preferably about 100 ul, of the color reagents are contacted wtih the antigen source.
- the slides are placed in a slide chamber and incubated for about 30 minutes at 55 °C. to permit formation of the formazan stain.
- the excess color reagent is then washed from the slide with PBS, soaked for about 15 minutes in cold PBS and the excess moisture again removed with the absorbent paper.
- the slide is then prepared for light microsoope viewing according to standard techniques. Briefly, a glycerol-gelatin mounting medium is placed in a 55°C. incubator for about 5 to 10 minutes for liquefication, and a large drop placed over the antigen source. The antigen source is then covered with a cover slip, taking care to avoid formation of air bubbles. When the slides are examined under a light microscope, the existence of antinuclear antibodies in the test serum will cause slightly enlarged, homo ⁇ geneous dark nuclei to appear, substantially without dark background staining.
- the present invention provides an ef ⁇ fective, inexpensive, highly visible, and permanent record of an antinuclear antibody assay. Further, the nuclear antigen source fixed onto the slide is extremely stable and does not require storage at low temperatures.
Abstract
For use in the immunohistochemical detection of antigens and related antibodies in a test serum, an antigen source fixed on a support and stabilized by dehydration treatment with an organic solvent mixture, such as aceton methanol, or mixtures thereof. The support is stored under nitrogen in a sealed polyethylene bag. Further, an immunoenzymatic method for the detection of biological components for a test sample, the method comprising the steps of: contacting the stabilized antigen source with the test sample, contacting the antigen source with an antibody conjugated with glucose oxidase and reactive with the component; incubating the antigen source with a solution containing glucose and a chromagenic mixture; and analyzing the antigen source for the presence of color, preferably with a light microscope.
Description
GLUCOSE OXIDASE IMMUNOHISTOCHEMICAL DETECTION OF ANTINUCLEAR ANTIBODIES
BACKGROUND OF THE INVENTION
This invention relates generally to immuno¬ enzymatic processes for use in medical diagnoses, and more particularly, to an immunohistochemical process for the detection of antinuclear antibodies in test serum. In recent years, an increasing amount of research has focused on improving clinical laboratory tests. A primary thrust of this research has been to replace the traditional radiotracer labels with safer and more stable labels.
The research efforts have been quite successful for a number of clinical assays. For example, in tests for antinuclear antibodies (ANA) , fluorescent markers have found wide acceptance. While fluorescent markers give quick results, they have not proven entirely satis- factory for a number of assays because once placed in a detectable state they have a very limited shelf life, fading after a few days or more. Moreover, fluorescent assays frequently exhibit high background due to auto-
* fluorescence caused by non-specific binding to various proteins, and are impractical for some laboratories because they require the use of relatively expensive fluorescent microscopes.
Another procedure for replacing radiotracer based assays, utilizing enzyme markers in immunoassay and immunohistochemical tests, has been practiced by a number of clinical laboratories in recent years. One of the most commonly used enzymes is horseradish peroxidase, which has been successfully used as a marker in immuno¬ histochemical procedures for the detection of a number of tissue antigens and related antibodies. Due to the relatively small size and high cellular penetration of this enzyme, it has found particular utility in the field of electron microscopy. For the light microscope, horseradish peroxidase has not proven entirely satis¬ factory, however, because of a high tendency to produce non-specific background stain, even in spite of attempts to quench the presence of endogenous peroxidase-like activity. Another objectionable feature of the peroxi¬ dase label is that it generally requires the use of a highly carcinogenic material, diaminobenzidine, for superior stain development. Although substitutes for this material have been developed, their contrast and intensity are typically not entirely satisfactory.
From the foregoing, it will be appreciated that there exists a definite need for a simple, inexpensive, safe and reliable antinuclear antibody detection method that can produce a relatively permanent record of the assay substantially in the absence of non-specific background The present invention fulfills this need.
SUMMARY OF THE INVENTION
The present invention provides an immunohisto- chemical method which substantially reduces the amount of background stain caused by endogenous materials, while producing a relatively permanent record of the assay.
Moreover, the reagents of the present invention are relatively inexpensive to manufacture, are stable for long periods of time even when stored at room tempera¬ ture, and have minimal carcinogenic characteristics.
In accordance with the invention, an immuno¬ enzymatic process for the detection of a component of a test sample is provided, said process comprising steps of: contacting the test sample with an antigen source fixed onto support and stabilized by dehydration; contacting the antigen source with an antibody conjugated with glucose oxidase and reactive with the component; incubating the antigen source with the solution containing glucose and a chro ogenic mixture and; analyzing the antigen source for the presence of color from the incubation.
The chromogenic mixtures preferably contain t-nitroblue tetrazolium chloride and m-phenazine metho- sulfate, which permit analysis with a light microscope. When the component of the test sample is an antinuclear antibody, the antigen source fixed on a glass slide is preferably Hep-2 cells or rat kidney tissue, and the conjugated antibody is goat immunoglobulin reactive with human immunoglobulins.
More particularly, an immunohistoche ical method for detecting antinuclear antibodies in a test serum, substantially in the absence of background stain¬ ing, is provided. This method comprises steps of: providing a nuclear antigen source fixed onto a slide and dehydrated with acetone at about 4*C;
uυ *- U '
contacting the nuclear antigen source with the test serum; contacting the nuclear antigen source with goat immunoglobulin conjugated with glucose oxidase and reactive with the .antinuclear antibodies; incubating the nuclear antigen source with a solution containing beta-D-glucose, t-nitroblue tetra- zolium, and 1-methoxyphenazine methosulfate; and analyzing the nuclear antigen source under a light microscope for the presence of a formazan stain.
Another aspect of the invention is a method of stabilizing an im unochemical substrate fixed on a solid support, the method comprising the steps of: providing a substrate fixed on the support; contacting the substrate with an organic solvent reagent to substantially dehydrate the substrate; placing the support into a protective sheath; backfilling the sheath with a gas; and sealing the support in the sheath.
The support is preferably a glass slide, and the substrate Hep-2 cells or rat kidney tissue. The organic solvent reagent can be composed of acetone, methanol, or mixtures thereof, and the interacting step performed between about 4 and 8° C. The sheath can be composed of a polyester film, and can contain a des- sicant. The preferred gas is nitrogen gas.
Other aspects and advantages of the present invention will become apparent from the following de¬ scription of the preferred embodiment, which disclose, by way of example, the principles of the invention.
:π
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Exemplary starting materials useful in practicing the present invention are slides fixed with human epethelial cells (Hep-2) or with rat kidney tissue suitable for use in antinuclear antibody assays. Such slides can be purchased commercially from a number of sources, including Antibodies Incorporated, Davis, California, and Behring, La Jolla, California, or, alternatively, can be manufactured according to con- ventional techniques. Generally, these slides should be stored at -20°C. to prevent degradation.
In accordance with the present invention, the slides are immersed' for about 10 minutes in a bath containing histological grade acetone, methanol, or a combination of these organic solvents. The bath is maintained at a temperature preferably ranging from about 4 C. to about 8 C. After removing the slides from the bath, they are allowed to air dry for about 2 to 3 minutes. Although the dehydration caused by treat- ent with the organic solvent mixture has significant¬ ly stabilized the substrate fixed on the slides, the slides are preferably stored at about 5°C. during continued processing.
After dehydration, the slides are placed into a protective sheath, preferably made from a poly¬ ethylene film, such as MYLAR bags with an aluminum outer layer. A small packet containing a standard dessicant is placed in the bag, and the bag is partially sealed. Nitrogen gas is then introduced through the unsealed portion of the bag until the bag bulges slightly. The bag is then totally sealed to prevent the nitrogen gas from escaping.
O PI
Goat immunoglobulin conjugated with the glucose oxidase enzyme can be prepared as follows. Glucose oxidase, which is available from a variety of commercial sources, including Sigma, St. Louis, Missouri, and Boehringer Manneheim, Indianapolis, Indiana, is dissolved in distilled water at a concentration of about 12 mg/ml. To this solution is added about 0.2 ml of ,0.1 M sodium periodate. The mixture is stirred for about 20 minutes and then dialyzed, preferably against 1 mM sodium acetate buffer, pH 4.4, for about 16 hours. After dialysis, the glucose oxidase solution is mixed with 1 ml of 0.01 M sodium bicarbonate buffer, pH 9.5, containing about 8 mg of the IgG fraction of goat anti- human im unoglobulins (IgG, IgA, and IgM) . After stir- ring for about two hours, 0.1 mis of. an aqueous solution containing about 4 mg/ml sodium borohydrate water solu¬ tion is added. When that reaction has completed (about 2 hours at 4 °C) , the entire mixture can be chromatographed on a gel filtration column, such as a Sephacryl S-300, available from Pharmacia, Piscataway, New Jersey, and the fractions containing the enzyme-IgG conjugate pooled. This pool can then be titrated. to determine working dilutions.
In accordance with another aspect of the invention, a method for detecting antinuclear antibodies utilizing these reagents is performed as follows. A small amount of test serum, or other test solution, is contacted with the nuclear antigen fixed source on the slide. After about thirty minutes at room temperature, the serum is washed from the slide with phosphate buf¬ fered saline (PBS) , and then soaked in a cold PBS solu¬ tion for about 10 minutes. The slides are removed from the PBS and excess moisture blotted with absorbent paper.
A small amount of the enzy e-IgG conjugate, preferably about 50 ul, is contacted with the antigen source for about 30 minutes at room temperature. The slide is again washed with PBS, soaked for 10 minutes in a cold PBS solution, removed, and excess moisture blotted with absorbent paper.
At this time, the color reagent may be pre¬ pared. It consists of about 2 volumes beta-D-glucose, 15 mg/ml in 0.1 M sodium phosphate buffer, pH 6.9, one volume t-nitroblue tatrazolium (t-NBT) , 2 mg/ml in the same buffer (if necessary, this may be filtered through Whattman No. 1 filter paper just prior to use) , and 1 volume of 1-methoxyphenazine methosulfate (m-PMS), 0.8 mg/ml in the same buffer. Although the preferred chro o- genic reagents are t-NBT and m-PMS, other materials can also be utilized. To ensure utarotation of the glucose molecules to their beta form, the mixture may be prepared about 1 hour before the staining reaction.
A small volume, preferably about 100 ul, of the color reagents are contacted wtih the antigen source. The slides are placed in a slide chamber and incubated for about 30 minutes at 55 °C. to permit formation of the formazan stain. The excess color reagent is then washed from the slide with PBS, soaked for about 15 minutes in cold PBS and the excess moisture again removed with the absorbent paper.
The slide is then prepared for light microsoope viewing according to standard techniques. Briefly, a glycerol-gelatin mounting medium is placed in a 55°C. incubator for about 5 to 10 minutes for liquefication, and a large drop placed over the antigen source. The antigen source is then covered with a cover slip, taking care to avoid formation of air bubbles.
When the slides are examined under a light microscope, the existence of antinuclear antibodies in the test serum will cause slightly enlarged, homo¬ geneous dark nuclei to appear, substantially without dark background staining.
From the foregoing description, it should be apparent that the present invention provides an ef¬ fective, inexpensive, highly visible, and permanent record of an antinuclear antibody assay. Further, the nuclear antigen source fixed onto the slide is extremely stable and does not require storage at low temperatures.
While a particular* form of the invention has been described in detail with reference to its presently preferred embodiment, it will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and scope of • the invention. Accordingly, it is not intended that the invention be limited except by the appended claims.
Claims
1. An immunoenzymatic process for the de¬ tection of a component of a test sample; said process comprising the steps of: contacting said test sample with an anti- gen source fixed onto a support and stabilized by dehy¬ dration; contacting the antigen source with an anti¬ body conjugated with glucose oxidase and reactive with said component; incubating the antigen source with a so¬ lution containing glucose and a chromogenic mixture; and analyzing the antigen source for the presence of color from the incubation.
2. The method of Claim 1, wherein the chrom- agenic mixture consists essentially of t-nitroblue tetrazolium chloride and m-phenazine ethosulf ate .
3. The method of Claim 1, wherein said an¬ tigen source is Hep-2 cells or rat kidney tissue.
4. The method of Claim 1, wherein said sup¬ port is a glass slide.
5. The method of Claim 1, wherein said con¬ jugated antibody is goat immunoglobulin reactive with human immunoglobulins.
6. The method of Claim 1, wherein said com¬ ponent is an antinuclear antibody.
7. The method of Claim 1, wherein said an¬ alysis is accomplished with a light microscope.
8. A method of stabilizing an immunochem- ical substrate fixed on a solid support, said method comprising the steps of: providing a substrate fixed on said sup¬ port; interacting said substrate with an organic solvent reagent to substantially dehydrate said substrate; placing said support into a protective sheath; backfilling said sheath with a gas; and sealing said support in said sheath.
9. The method of Claim 8, wherein said sup¬ port is a glass slide.
10. The method of Claim 8, wherein said substrate consists essentially of Hep-2 cells.
11. The method of Claim 8, wherein said organic solvent reagent is acetone, methanol or mixtures thereof.
12. The method of Claim 8, wherein said contacting step is performed a,t about -between 4 and 8° C.
13. The method of Claim 8, wherein said sheath is a polyester film.
14. The method of Claim 8, wherein said sheath contains a dessicant.
15. The method of Claim 8, wherein said gas is nitrogen gas.
16. An immunohistoche ical method for de¬ tecting antinuclear antibodies in a test serum sub¬ stantially in the absence of background staining, said method comprising the steps of: providing a nuclear antigen source fixed onto a slide and dehydrated with acetone at about 4° C. ; contacting the nuclear antigen source with said test serum; contacting the nuclear antigen source with goat immunoglobulin conjugated with glucose oxidase and reactive with said antinuclear antibodies; incubating said nuclear antigen source with a solution containing beta-D-glucose, t-nitroblu-e tetrazolium, and 1-methoxyphenazine methosulfate; and analyzing said nuclear antigen source under a light microscope for the presence of a formazan stain.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU89546/82A AU8954682A (en) | 1981-08-31 | 1982-08-12 | Glucose oxidase immunohistochemical detection of antinuclear antibodies |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US29811281A | 1981-08-31 | 1981-08-31 | |
US298,112810831 | 1981-08-31 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1983000877A1 true WO1983000877A1 (en) | 1983-03-17 |
Family
ID=23149085
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1982/001107 WO1983000877A1 (en) | 1981-08-31 | 1982-08-12 | Glucose oxidase immunohistochemical detection of antinuclear antibodies |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0091911A4 (en) |
CA (1) | CA1206092A (en) |
ES (2) | ES515350A0 (en) |
IT (1) | IT1153188B (en) |
WO (1) | WO1983000877A1 (en) |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0094603A1 (en) * | 1982-05-14 | 1983-11-23 | American Hoechst Corporation | Enzyme/immunofluorescent assay for autoantibodies |
US4588682A (en) * | 1982-12-13 | 1986-05-13 | Integrated Genetics, Inc. | Binding nucleic acid to a support |
EP0206779A1 (en) * | 1985-06-21 | 1986-12-30 | Modern Diagnostics, Inc. | Detecting antinuclear antibody |
EP0241025A1 (en) * | 1986-04-08 | 1987-10-14 | Whittaker Bioproducts, Inc. | Method and substrate for immunofluorescent microscopy |
WO1988009932A1 (en) * | 1987-06-03 | 1988-12-15 | Amrad Corporation Limited | NUCLEAR ANTIGEN La |
US4814269A (en) * | 1985-03-29 | 1989-03-21 | Cenfold Holdings S.A. | Diagnostic testing for antibodies against microorganisms |
EP0335293A2 (en) * | 1988-03-30 | 1989-10-04 | Hoechst Japan Limited | A method of preparing an analytical element for the determination of anti-nuclear antibodies |
WO2005079814A1 (en) * | 2004-02-20 | 2005-09-01 | Beijing Xinjing Antai Medical And Technology Service Limited Corp. | A pharmaceutical composition used for treating recurrent spontaneous abortion and method thereof |
WO2005080980A1 (en) * | 2004-02-20 | 2005-09-01 | Beijing Xinjing Antai Medical And Technology Service Limited Corp. | A method for diagnosing immunity recurrent spontaneous abortion and method for treating and monitoring |
US20100240551A1 (en) * | 2005-07-05 | 2010-09-23 | Cytoskeleton, Inc. | Detection Of Rho Proteins |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3146163A (en) * | 1962-01-23 | 1964-08-25 | John H Brewer | Apparatus for separating certain components from blood |
US3791932A (en) * | 1971-02-10 | 1974-02-12 | Akzona Inc | Process for the demonstration and determination of reaction components having specific binding affinity for each other |
US3876504A (en) * | 1974-06-10 | 1975-04-08 | Early Warning Co | Procedure for determination of antigens and antibodies and articles for use therewith |
US3979509A (en) * | 1974-09-03 | 1976-09-07 | General Electric Company | Opaque layer method for detecting biological particles |
US3997657A (en) * | 1973-10-15 | 1976-12-14 | American Cyanamid Company | Dry slide reagent employed in immunofluorescent test for detection of human antinuclear factor |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2836362A1 (en) * | 1978-08-19 | 1980-03-06 | Behringwerke Ag | DIAGNOSTIC MEANS |
-
1982
- 1982-08-12 WO PCT/US1982/001107 patent/WO1983000877A1/en not_active Application Discontinuation
- 1982-08-12 EP EP19820902930 patent/EP0091911A4/en not_active Withdrawn
- 1982-08-30 ES ES515350A patent/ES515350A0/en active Granted
- 1982-08-30 CA CA000410409A patent/CA1206092A/en not_active Expired
- 1982-08-31 IT IT23075/82A patent/IT1153188B/en active
-
1983
- 1983-07-16 ES ES524183A patent/ES8500999A1/en not_active Expired
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3146163A (en) * | 1962-01-23 | 1964-08-25 | John H Brewer | Apparatus for separating certain components from blood |
US3791932A (en) * | 1971-02-10 | 1974-02-12 | Akzona Inc | Process for the demonstration and determination of reaction components having specific binding affinity for each other |
US3997657A (en) * | 1973-10-15 | 1976-12-14 | American Cyanamid Company | Dry slide reagent employed in immunofluorescent test for detection of human antinuclear factor |
US3876504A (en) * | 1974-06-10 | 1975-04-08 | Early Warning Co | Procedure for determination of antigens and antibodies and articles for use therewith |
US3979509A (en) * | 1974-09-03 | 1976-09-07 | General Electric Company | Opaque layer method for detecting biological particles |
Non-Patent Citations (4)
Title |
---|
Am. J. Clin. Path., Vol. 71, issued May 1979, S.C. SUFFIN et al, "Improvement ofthe Glucose Oxidase Immunoenzyme Technic". * |
Clin. Chim. Acta, Vol. 101, issued 1979, NAKAMURA et al, "Use of 1-Methoxy-5- Methylphenazinium Methyl Sulfate (1-Methoxy Pms) is the Assay of some Enzymes of Diagnostic Importance". * |
J. Histochem. Cytochem., Vol. 17, No. 9, issued 1969, T.E. MASON et al, "An Immunoglobulin-Enzyme Bridge Method For Localizing Tissue Antigens". * |
J. Immunol. Meth., Vol 39, issued 1980, J.GUESDON et al, "Lectin Immuno Tests: Quantitation and Titration of Antigens and Antibodies Using Lectin-Antibody Conjugates". * |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4487830A (en) * | 1982-05-14 | 1984-12-11 | American Hoechst Corporation | Enzyme/immunofluorescent assay for autoantibodies |
EP0094603A1 (en) * | 1982-05-14 | 1983-11-23 | American Hoechst Corporation | Enzyme/immunofluorescent assay for autoantibodies |
US4588682A (en) * | 1982-12-13 | 1986-05-13 | Integrated Genetics, Inc. | Binding nucleic acid to a support |
US4814269A (en) * | 1985-03-29 | 1989-03-21 | Cenfold Holdings S.A. | Diagnostic testing for antibodies against microorganisms |
EP0206779A1 (en) * | 1985-06-21 | 1986-12-30 | Modern Diagnostics, Inc. | Detecting antinuclear antibody |
EP0241025A1 (en) * | 1986-04-08 | 1987-10-14 | Whittaker Bioproducts, Inc. | Method and substrate for immunofluorescent microscopy |
WO1988009932A1 (en) * | 1987-06-03 | 1988-12-15 | Amrad Corporation Limited | NUCLEAR ANTIGEN La |
EP0335293A2 (en) * | 1988-03-30 | 1989-10-04 | Hoechst Japan Limited | A method of preparing an analytical element for the determination of anti-nuclear antibodies |
EP0335293A3 (en) * | 1988-03-30 | 1990-09-12 | Hoechst Japan Limited | A method of preparing an analytical element for the determination of anti-nuclear antibodies |
WO2005079814A1 (en) * | 2004-02-20 | 2005-09-01 | Beijing Xinjing Antai Medical And Technology Service Limited Corp. | A pharmaceutical composition used for treating recurrent spontaneous abortion and method thereof |
WO2005080980A1 (en) * | 2004-02-20 | 2005-09-01 | Beijing Xinjing Antai Medical And Technology Service Limited Corp. | A method for diagnosing immunity recurrent spontaneous abortion and method for treating and monitoring |
US7674590B2 (en) | 2004-02-20 | 2010-03-09 | Beijing Xinjing Antai Medical And Technology Service Limited Corp. | Methods for diagnosing and for monitoring the treatment of recurrent spontaneous abortion |
US7902162B2 (en) | 2004-02-20 | 2011-03-08 | Beijin Xinjing Antai Medical and Technology Service Limited Corp | Medicament and method for treating recurrent spontaneous abortion |
US20100240551A1 (en) * | 2005-07-05 | 2010-09-23 | Cytoskeleton, Inc. | Detection Of Rho Proteins |
Also Published As
Publication number | Publication date |
---|---|
ES524183A0 (en) | 1984-11-01 |
ES8400773A1 (en) | 1983-11-01 |
EP0091911A4 (en) | 1984-04-06 |
IT1153188B (en) | 1987-01-14 |
EP0091911A1 (en) | 1983-10-26 |
ES515350A0 (en) | 1983-11-01 |
CA1206092A (en) | 1986-06-17 |
IT8223075A0 (en) | 1982-08-31 |
ES8500999A1 (en) | 1984-11-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US4487830A (en) | Enzyme/immunofluorescent assay for autoantibodies | |
US4497899A (en) | Immunoassay for Chlamydia trachomatis antigens | |
Mason et al. | Alkaline phosphatase and peroxidase for double immunoenzymatic labelling of cellular constituents. | |
US4690907A (en) | Capillary tube immunoassay | |
US4469797A (en) | Digoxigenin immunogens, antibodies, labeled conjugates, and related derivatives | |
US4302534A (en) | Chemiluminescent enzyme immunoassay | |
US4474878A (en) | Sandwich EIA for antigen associated with hepatitis | |
JPH05506930A (en) | Method for measuring analytes in biological fluids in the presence of substances that interfere with the analyte assay | |
US5312744A (en) | Method of immobilizing and preserving an immunologically reactive substance | |
CA1210691A (en) | Determination of terminal deoxynucleotidyl transferase | |
IE48400B1 (en) | Process for immunologic determination tests | |
US4642285A (en) | Sandwich EIA for antigen | |
IE50457B1 (en) | Method for detecting and determining proteinaceous specific binding agents and materials bindable thereto,test composition and test kit therefor | |
US5187066A (en) | Methods for detecting amphiphilic antigens | |
WO1983000877A1 (en) | Glucose oxidase immunohistochemical detection of antinuclear antibodies | |
JPH0116388B2 (en) | ||
US4837170A (en) | Monoclonal antibody, measuring method of glucosylated protein by utilizing the monoclonal antibody, and kit therefor | |
US4729961A (en) | Process for the detection and assay by erythroadsorption | |
CA1104927A (en) | Diagnostic immunochemical test material and procedure | |
CA1302244C (en) | Dry test strips having a red blood cell exclusion layer preventing interference by red blood cells in analyte detection visualization | |
Lebeau et al. | Histochemistry and immunohistochemistry on bone marrow biopsies: a rapid procedure for methyl methacrylate embedding | |
KAMEYA et al. | Enzyme-and immuno-histochemical localization of human placental alkaline phosphatase | |
Aarli | Localization and Properties of an Acid‐Solubl'e Muscle Antigen Reacting with Antibodies in Myasthenia Gravis Sera | |
Van Noorden et al. | Immunocytochemistry | |
US4460694A (en) | Bovine glycoproteins and use in diagnosing infectious mononucleosis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Designated state(s): AU JP |
|
AL | Designated countries for regional patents |
Designated state(s): AT BE CH DE FR GB NL |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1982902930 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1982902930 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1982902930 Country of ref document: EP |