CA2060377A1 - Determination of high molecular weight analytes - Google Patents
Determination of high molecular weight analytesInfo
- Publication number
- CA2060377A1 CA2060377A1 CA002060377A CA2060377A CA2060377A1 CA 2060377 A1 CA2060377 A1 CA 2060377A1 CA 002060377 A CA002060377 A CA 002060377A CA 2060377 A CA2060377 A CA 2060377A CA 2060377 A1 CA2060377 A1 CA 2060377A1
- Authority
- CA
- Canada
- Prior art keywords
- analyte
- enzyme
- complex
- antibody
- binding moiety
- 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
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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
-
- 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
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
Abstract
DETERMINATION OF HIGH MOLECULAR WEIGHT ANALYTES
ABSTRACT
A method for detecting the presence of a high-molecular-weight analyte, which uses a variation on complementation assays, comprising (1) providing an enzyme donor (ED)complex comprising ED coupled to an analyte-specific binding molecules wherein the ED
complex retains measurable complementation activity such that active .beta.-galactosodase formed in the presence of an enzyme acceptor (EA); (2) contacting the ED complex and EA in the presence of a sample suspected of containing a high-molecular-weight analyte that reacts specifically with the analyte specific binding molecule; and (3) relating the presence of the analyte in the sample to the formation of active .beta.-galactosidase enzyme.
ABSTRACT
A method for detecting the presence of a high-molecular-weight analyte, which uses a variation on complementation assays, comprising (1) providing an enzyme donor (ED)complex comprising ED coupled to an analyte-specific binding molecules wherein the ED
complex retains measurable complementation activity such that active .beta.-galactosodase formed in the presence of an enzyme acceptor (EA); (2) contacting the ED complex and EA in the presence of a sample suspected of containing a high-molecular-weight analyte that reacts specifically with the analyte specific binding molecule; and (3) relating the presence of the analyte in the sample to the formation of active .beta.-galactosidase enzyme.
Description
s~
MCRO-~22/00 US
D~T~NIN~TION OF HI~ NOL~CUL~R N~I~T ~NAL~T~S
INTRODUCT ON
Technical Field The pre~ent invention relates to enzyma immunoassays and, in particular, to immunoassay~ u~ing ~-galacto idase as the enzyme label.
Back~round Enzymes have been u~ed successfully as detectable labelg ln a numbsr of difPeren immuno-a~say~. A number of ~uch a~ay~ have be~n based on the ability of frayment~ of p-galacto~ida~e ~o complement each oth~r an~ form activ~ enzyme. In parti~ular, a p-~alactosida e enzyme donor (~D~, which i8 a fra~ment from one end of the en~yme molecule, combines with a p galacto~ida~e enzyme acceptor ~A)~
a fxa$ment from the other end of the enzyme molecule, to form ac~ive p-galactosida~e enz~ma. Con~ugatîng a small analyte or an analyte analogue to the ED at certain ~ites does not greatly affect the comple-mentation of ~D with EA or the rate of ~-galacto-sida3e-catalyzed activity. However, when the ED
analyte con~ugate i8 bound by anti-anslyte antibody, complementation and the enzyme-catalyzed reaction ra~e during the initial pha~e of th~ reaction are reduced.
This reduction in enzyme-catalyzed reaction rate has been u~ed to quantify analytes in a situation whare both the ED-analyte con~u~ate pr2sent in an assay mediu~ and the analyte pre~ent ~n the ~ample compete for anti-analyte antibody prior to the additlon of EA. The ~-galacto~ida~e-catalyzed reaction rate increa~es as the amount of analyte present in the sample increase~, because analyte in the sampl~ reduce~ interaction of the ED-analyte con~ugate and anti-analyte antlbody, allowing more of the ED-analyte con~ugate to react wlth the EA ~o form active ~-galactosida~e Pnzyme.
This technology wa~ originally developed :Eor low molecul~r weight analyte~. Recent developments in ~-galactosidase complementation a~ay~ have included new EA and ED molecules in which the break in continuity of the Q-galacto~i~a e chain i~ in the omega region at th~ carboxyl terminus of the chain in3tead of in the alpha resion at tho amino terminus, as wa~ or~ginally developed, as well as application of the technology to new assay t~pe~.
A problem exi~ts in as~ays for laxge molecular weight analyte~. In uch a~ay~ there i~
only a small relative efect on complementation rate when a binding partner bind~ to the large analyte or analyts analogue attached to the ED moleculs. Thi~ i~
not a problem with ~mall analyte~, a~ the attachment of a large binding protein (u~ually an antibody ~pecific for the anslyte) to the small analyte in such an ED-analyte con~ugate cau~e~ a significant decrea~e in complementation actlvity (i.e., the rate at which active ~-galactosida~e i~ formed fro~ the inactive ED
and EA fra~ments). However, when ~ large analyte i~
already attached to the ED fragment, attachment of an antibody molecule or other binding partner to the large molecule make~ little difference in the rate of complementation.
Accordingly, there remain~ a need for a new method capable of measuring high molecular weight analyte~ u~ing complementation of ED and EA fragment~
of ~-galactosidase that has increased sen~itivity over exi~ting methods.
Relevant Literature ~ odified ~-galactosidase enzyme donor~ and enzyme acceptor~ have bean prepared by chemical synthesi~ and recombinant DNA engineering. The 3 ~ V~
modified fragments retain ~-yalactosida3e activity upon complementation. See, for ex~mple, U.S. Patant No. 4,708,929 and the article~ cited therein. ~utant polyp~ptides derived fro~ ~-galacto~ida~e are di~closed by Langley and Zabinr Bio. Chem. (1976) 15:4866, which can complement or spontaneou~ly restore enzyme activity when added to extracts of appropriate p-galacto~idase-negative mutant~. Sea al~o Lin et at., Bio. Chem. Bio~h~. Ra~. Co~m. (1970) 40:249.
SUMMARY OF THE INVENTION
It i~ an ob~ect of thi~ invention to provide a method and rea~ents for the detection and quantitative analysis t using homogenou~ immunoa~ay sy~tem, of high molecular weight analyt~ that ha~
increa~ed 8en8itivity and ~i~plicity ov~r ~echnique~s previou31y avsilabla.
The pre3ent invention provide~ such a method and reagents by utilizing p-gal~cto~ida~e donor ~nd accep~or fragmants in a new assay format.
Specifically, the presence or quantity of a high-molecular-weight analyte in a ~ample i~ determined by employing compl~ment~ry fragment~ of p-galacto~ida~e, which fragment~ are defined a~ enzyme donor (~D) ancl en~yme acceptor (EA) and when bound, i.e., brought together, orm active p-galactosida~e, wherein the ED
frayment is pre~ent in the form of an ED/~pecific-binding-molecule con~ugate where the binding molecule, typically an antibody ~Ab), i8 specific for the analyt~. It has been found that sufficient complementation activity remain~ for an ED-Ab con~ugate (thi~ abbreviation being use as shorthand to de~ignate all pos~ible complexes of the invention that contain different types of binding molecules). Thi~
ED-Ab complementation activity i8 significantly affected when bound by the large analyte to be mea~ured. In general, the complementation activity of the Ab-ED con~ugate decrease~ in proportion to the f~
amount of ~D-Ab con~u~te bound by the an~lyte. Thu~, lt ~ posslble to determine the presence and amount of a high-molecular-weight an~lyteO Thi~ i~ in contra3t to prior assays which relied on competitive ~ntibody binding to a ~mall analyte~D conjugate to reduce complementation activity.
The re~ulting enzyme activity can be detected or measured in a variety of well known manners, such as by u~ing an enzyme ~ub~trate which provides a mea~urable product upon reaction with p~galactosidase. The amount of analyte present in a sample can thereby be determined by comparing the amount of measurable product to that formed when using a serie~ of samples containing known amount~ of analyte.
BRIE DESCRIP~ION OF THE D~AWINGS
The pre~ent invention will be better understood by referencQ to the following detailed description of the invention when con~idered in combination with the accompanying drawingR ~ wherein:
Figure 1 i8 a graph showing enzyme activity following complementation in ~he presence of a high-molecular-weight analyte (ferritin) by as~ay procedure 1 of Exampl~ 1 as de~çribed herein.
Figurs 2 i~ ~ graph showing enzyme activity following complemen~ation in the pre-~ence of low concentration~ of a high molecular-weight analy~e (ferritin~, thereby ~howing a~say sensitivity by as3~y procedure 1.
Figure 3 i8 a graph showing correlation between an assay of the invention and an FDA approved a~ay for ferritin by a~say procedure l.
Figure 4 is a graph showing snzyme activity following complementation in the presence of a high-molecular-weight analyte ~ferritin) by a~ay procedure 2 of Example 1 aR described herein.
Figure 5 i8 a ~raph 0howin~ corr~l~tion between an a~3sy of the invention and an FDA-approve~
as~ay for ferritin by a~8ay proeedure 2.
Flgure 6 i~ a graph showing enzyme activity following complementation in the pres~nca of C-reactive protein as de~cribed in the procedure for Ex~mple 2 a~ described herein.
Figure 7 i~ a graph ~howing correlation between an as~ay of the invention and an FDA-approved assay f~r C-reactive prot~in as de~cribed in the proceduxe for Example 2.
DESCRIPTION OF SP~CIFIC EMBODIMENTS
The pres~nt invention comprise~ method~ and reagent~ for detecting and quantitating the amount of a high-molecular-weight ~nalyte in a 8ampl The method i~ a variation on standard complem~ntatlon a~says involving p-galactosidaae in which inactlve fragment~ of p-gal~ctosid~ combine in ~olution ~o form active p-galacto~ida~e. The fragments are referre~ to hsrein ~ enzyme-donor (ED) and enzyme-acceptor (EA) fragmenta~ to designate the ~maller and larger fragments, re~pective~y, of the p-qalactosida~
molecule.
In particular, the present invent~on pro~id~s ~ method for dstecting the pre~ence of an analyte having a high molecular weight tpreferably with multivalent anti~enic determinants) such that complementation activity i~ different in the pre~ence and ab.~ence of binding between (1) antibody (Ab) or another bindin~ moiety attached to an enzyme donor fragment of p-galactosida~e and (2) the analyte. The rate of enzyme complementation i8 further reduced in the ca~e of multivalent analytes by the addition of excess unlabelled binding moiety, which effectively increase3 the ~ize of the parent analyte by binding to unoccupied binding ~ite~. In general, the method compri~es the following steps:
MCRO-~22/00 US
D~T~NIN~TION OF HI~ NOL~CUL~R N~I~T ~NAL~T~S
INTRODUCT ON
Technical Field The pre~ent invention relates to enzyma immunoassays and, in particular, to immunoassay~ u~ing ~-galacto idase as the enzyme label.
Back~round Enzymes have been u~ed successfully as detectable labelg ln a numbsr of difPeren immuno-a~say~. A number of ~uch a~ay~ have be~n based on the ability of frayment~ of p-galacto~ida~e ~o complement each oth~r an~ form activ~ enzyme. In parti~ular, a p-~alactosida e enzyme donor (~D~, which i8 a fra~ment from one end of the en~yme molecule, combines with a p galacto~ida~e enzyme acceptor ~A)~
a fxa$ment from the other end of the enzyme molecule, to form ac~ive p-galactosida~e enz~ma. Con~ugatîng a small analyte or an analyte analogue to the ED at certain ~ites does not greatly affect the comple-mentation of ~D with EA or the rate of ~-galacto-sida3e-catalyzed activity. However, when the ED
analyte con~ugate i8 bound by anti-anslyte antibody, complementation and the enzyme-catalyzed reaction ra~e during the initial pha~e of th~ reaction are reduced.
This reduction in enzyme-catalyzed reaction rate has been u~ed to quantify analytes in a situation whare both the ED-analyte con~u~ate pr2sent in an assay mediu~ and the analyte pre~ent ~n the ~ample compete for anti-analyte antibody prior to the additlon of EA. The ~-galacto~ida~e-catalyzed reaction rate increa~es as the amount of analyte present in the sample increase~, because analyte in the sampl~ reduce~ interaction of the ED-analyte con~ugate and anti-analyte antlbody, allowing more of the ED-analyte con~ugate to react wlth the EA ~o form active ~-galactosida~e Pnzyme.
This technology wa~ originally developed :Eor low molecul~r weight analyte~. Recent developments in ~-galactosidase complementation a~ay~ have included new EA and ED molecules in which the break in continuity of the Q-galacto~i~a e chain i~ in the omega region at th~ carboxyl terminus of the chain in3tead of in the alpha resion at tho amino terminus, as wa~ or~ginally developed, as well as application of the technology to new assay t~pe~.
A problem exi~ts in as~ays for laxge molecular weight analyte~. In uch a~ay~ there i~
only a small relative efect on complementation rate when a binding partner bind~ to the large analyte or analyts analogue attached to the ED moleculs. Thi~ i~
not a problem with ~mall analyte~, a~ the attachment of a large binding protein (u~ually an antibody ~pecific for the anslyte) to the small analyte in such an ED-analyte con~ugate cau~e~ a significant decrea~e in complementation actlvity (i.e., the rate at which active ~-galactosida~e i~ formed fro~ the inactive ED
and EA fra~ments). However, when ~ large analyte i~
already attached to the ED fragment, attachment of an antibody molecule or other binding partner to the large molecule make~ little difference in the rate of complementation.
Accordingly, there remain~ a need for a new method capable of measuring high molecular weight analyte~ u~ing complementation of ED and EA fragment~
of ~-galactosidase that has increased sen~itivity over exi~ting methods.
Relevant Literature ~ odified ~-galactosidase enzyme donor~ and enzyme acceptor~ have bean prepared by chemical synthesi~ and recombinant DNA engineering. The 3 ~ V~
modified fragments retain ~-yalactosida3e activity upon complementation. See, for ex~mple, U.S. Patant No. 4,708,929 and the article~ cited therein. ~utant polyp~ptides derived fro~ ~-galacto~ida~e are di~closed by Langley and Zabinr Bio. Chem. (1976) 15:4866, which can complement or spontaneou~ly restore enzyme activity when added to extracts of appropriate p-galacto~idase-negative mutant~. Sea al~o Lin et at., Bio. Chem. Bio~h~. Ra~. Co~m. (1970) 40:249.
SUMMARY OF THE INVENTION
It i~ an ob~ect of thi~ invention to provide a method and rea~ents for the detection and quantitative analysis t using homogenou~ immunoa~ay sy~tem, of high molecular weight analyt~ that ha~
increa~ed 8en8itivity and ~i~plicity ov~r ~echnique~s previou31y avsilabla.
The pre3ent invention provide~ such a method and reagents by utilizing p-gal~cto~ida~e donor ~nd accep~or fragmants in a new assay format.
Specifically, the presence or quantity of a high-molecular-weight analyte in a ~ample i~ determined by employing compl~ment~ry fragment~ of p-galacto~ida~e, which fragment~ are defined a~ enzyme donor (~D) ancl en~yme acceptor (EA) and when bound, i.e., brought together, orm active p-galactosida~e, wherein the ED
frayment is pre~ent in the form of an ED/~pecific-binding-molecule con~ugate where the binding molecule, typically an antibody ~Ab), i8 specific for the analyt~. It has been found that sufficient complementation activity remain~ for an ED-Ab con~ugate (thi~ abbreviation being use as shorthand to de~ignate all pos~ible complexes of the invention that contain different types of binding molecules). Thi~
ED-Ab complementation activity i8 significantly affected when bound by the large analyte to be mea~ured. In general, the complementation activity of the Ab-ED con~ugate decrease~ in proportion to the f~
amount of ~D-Ab con~u~te bound by the an~lyte. Thu~, lt ~ posslble to determine the presence and amount of a high-molecular-weight an~lyteO Thi~ i~ in contra3t to prior assays which relied on competitive ~ntibody binding to a ~mall analyte~D conjugate to reduce complementation activity.
The re~ulting enzyme activity can be detected or measured in a variety of well known manners, such as by u~ing an enzyme ~ub~trate which provides a mea~urable product upon reaction with p~galactosidase. The amount of analyte present in a sample can thereby be determined by comparing the amount of measurable product to that formed when using a serie~ of samples containing known amount~ of analyte.
BRIE DESCRIP~ION OF THE D~AWINGS
The pre~ent invention will be better understood by referencQ to the following detailed description of the invention when con~idered in combination with the accompanying drawingR ~ wherein:
Figure 1 i8 a graph showing enzyme activity following complementation in ~he presence of a high-molecular-weight analyte (ferritin) by as~ay procedure 1 of Exampl~ 1 as de~çribed herein.
Figurs 2 i~ ~ graph showing enzyme activity following complemen~ation in the pre-~ence of low concentration~ of a high molecular-weight analy~e (ferritin~, thereby ~howing a~say sensitivity by as3~y procedure 1.
Figure 3 i8 a graph showing correlation between an assay of the invention and an FDA approved a~ay for ferritin by a~say procedure l.
Figure 4 is a graph showing snzyme activity following complementation in the presence of a high-molecular-weight analyte ~ferritin) by a~ay procedure 2 of Example 1 aR described herein.
Figure 5 i8 a ~raph 0howin~ corr~l~tion between an a~3sy of the invention and an FDA-approve~
as~ay for ferritin by a~8ay proeedure 2.
Flgure 6 i~ a graph showing enzyme activity following complementation in the pres~nca of C-reactive protein as de~cribed in the procedure for Ex~mple 2 a~ described herein.
Figure 7 i~ a graph ~howing correlation between an as~ay of the invention and an FDA-approved assay f~r C-reactive prot~in as de~cribed in the proceduxe for Example 2.
DESCRIPTION OF SP~CIFIC EMBODIMENTS
The pres~nt invention comprise~ method~ and reagent~ for detecting and quantitating the amount of a high-molecular-weight ~nalyte in a 8ampl The method i~ a variation on standard complem~ntatlon a~says involving p-galactosidaae in which inactlve fragment~ of p-gal~ctosid~ combine in ~olution ~o form active p-galacto~ida~e. The fragments are referre~ to hsrein ~ enzyme-donor (ED) and enzyme-acceptor (EA) fragmenta~ to designate the ~maller and larger fragments, re~pective~y, of the p-qalactosida~
molecule.
In particular, the present invent~on pro~id~s ~ method for dstecting the pre~ence of an analyte having a high molecular weight tpreferably with multivalent anti~enic determinants) such that complementation activity i~ different in the pre~ence and ab.~ence of binding between (1) antibody (Ab) or another bindin~ moiety attached to an enzyme donor fragment of p-galactosida~e and (2) the analyte. The rate of enzyme complementation i8 further reduced in the ca~e of multivalent analytes by the addition of excess unlabelled binding moiety, which effectively increase3 the ~ize of the parent analyte by binding to unoccupied binding ~ite~. In general, the method compri~es the following steps:
2 ~ ~ ~ 3 ~ 1 ~ a) providing an ~D complex compri~1ng ED
coupled to an analyte-specific bindlng molecula, wherein said ED complex retain~ mes~urable complementation activity ~uch that active ~-~alactosida~e i~ formed in the presence of EA;
(b) contacting th~ ED complex and E~ (andan exc~s~ unlabelled binding moiety in preferred embodiments u ing ~ultivalen~ analyte~) in ~he pre~ence of a aample 3uspected of containing a high~
molecular-weight analyte that react~ ~peciically wi~h the analyte-~pecific binding molecule, and (c) relating the pres~nce of the analyte in the sample to the fonmation of active ~-galacto~idase enzyme.
lS The present invention ~ predicated, at least in part, on the principle that the ~D/bindiny-moiety complex and EA for~ actLve enz~ne at a differont rate than does the combination of E~ with the ternary complex of ~D/binding-moiety/high-molecular-weight analyte. When a ~m 11 amount of the analyts i~ pre~ent, only a part o~ ED-Ab con~uga~e can bind to the analyte and hence more of ED-Ab con~ugate i~ available for complem~ntation with EA. To the contrary, when large amount~ of analyte are pre~ent, le~s ED-~b coniugate i~ available for complementstion with EA, and hencs, les~ enzyme activity i3 ob~er~ed.
In both case3, multivalent antigenic determinants allow the s~multaneous complexation of ED-binding moiety con~ugate and unla~elled anti-analyte antibody binding at multiple ~ite~. Unlabelled antibody occupie3 the unbound 8ite8 and makes enzyme complementation inhibition more effective. The ~ize I of the ternary complex, consi~ting of ED-~h con~ugate, analyte, snd unlabelled antibody become~
very large per ED molecule, and hence les3 complementation i3 observed. The quanti~y of the analyte, therefore, i~ in~ersely proportional to h~3 ~
enzyms activity, which c~n be measured in any ~uitable manner, as through the u~e of an enzyme ~ub~ra~e.
The present inventive method therefore further contemp}ates the ED-~b complex t EA~ unlabelled ant~body, and sample being contacted with an enz~me sub~trate which provides or a mea~ur~ble product upon reaction with complementad p-galactosida~e such that the amount of analyte in the ~ample can be de~enmined by comparing the amount of measurable product to that formed in th~ presence of a kn~wn amount of analyte.
The pre~ent invention al80 contemplates kit~ for use in carrying out the method of the pre~ent invention as well as individual reagent~ used in the method of the invention.
The ability of ths ED-~b complex of the present invention to complement with EA in the absence of analyte differ~ markedly from ~he p-galactosidase complementation phenomenon observed in a number of prev$ou~1y described assays employing ED and E~
con~ugatea. For example, in the a~saya describ~d in US Patent 4,708,929, ED con~ugated to a small analyte complement~ with EA in the absence of anti-analyte antibodie3. ~hen the anti-analyte antibodiex are pre~ent in the assay mixture, complementation activity drops ~ignificantly when compared to initial activity in the ~bsence of antibodies. In the pxe~ence of analyte present in the unknown sample, a competition occurs between the ~D-labelled analyte and the analyte present in the sample, for a limited amount of binding sites. Thux, pre~ence of more analyte ties up more antibody and hence, allows corre4pondingly more ED-analyte to complement with EA. T}lus, complemented enzyme ~ctivity i8 directly proportional to the amount of analyte in the sample. Thi~ competitive assay method works well only with ~mall molecules.
In contra~t, the present invention i8 a non-competitive l~munoas~ay. An ED-binding moiety con~ugate is employed as compared to ED-hapten 3 ~ ~
con~ugate in the previously described method.
Further, the pre~eng method allow~ a direct assay where the analyte to be measured i~ u~ed to inhibit the rate of enzyme complementation. In addition, the present as~ay principle works well with high molecular weight analyte~ preferably having mult:L~alent antig&nic determinant~.
The difference in the prior teaching and the pre ent re8ult8 i~ believed to result from ~he large size of the analyte/ED-Ab~unlabelled Ab complex relati~e to the bLnding moietie~. By using the present a ~ay only when the analytes are very large, it i~ 8till po~sible to maint~in a difference in complementation rate~. Such difference~ would have been undetectable on ~he ~cal* of complementat$on rates known previously for prior as~ays in which ~mall analytes were att~ched to ED molecule~ and allowed to react with antibodies to reduce the complementation rate.
The ~ and EA componant~ of the pre~en~
invention are partia} sequences of p-galacto~idase.
For the purpos~ of the sub~ect invention, the shorter portion of the p-galactosidase molecule i~ referred to a~ the enzyme donor (~D), and the longer portion i~
the enzyme acceptor ~EA). The enzyme acceptor and enzyme donor are character$zed by forming an active enzyme complex when brought together. The preparation of ~-galacto~idase enzyme donor~ and acceptor~ is described in U.S. Patent No. 4,708,929, which disclo~ure i~ incorporated herein by reference.
Any of the previously described methods for attaching proteins to other molecules can be u~ed to attach the ED component to the binding mo~ety.
Especially preferred i8 the u3a of bifunctionsl organic linking group~ to attach proteinaceou~ binding moietie~ to the ED fragm~nt, which it~el~ i8 proteinaceou~. Example-q of such linking groups, which are well known in protein chemi~try, include 7~
dialdehyd4~, ~uch a~ glut~ral~ehyde, and diamlne~, such aR 1, 6-dlaminohexane as described in the ~pecif ic literature (e.g., J. In~nunoa~say, 1983, 4~209).
The binding moieties are components capable of 5 binding to t}le particular analyl:e of intere~t. The binding between the bind$n~ moietie~ and the an~lyte i~ preferably non-coval~nt. 5uitable binding moietie~
preferably have higher affin~ty and ~pecificity for the analyte than for the other components in a ~ample 10 being analyzed. Suitabla binding moieties can be of a variety of molecular categorie~ includlng antibodie~
specific for a portion of the analyte ~prefexably, monoclonal antibodie~ ); binding protein~ that naturally blnd to the analyte , e . S~ ., lectins iEor 15 analytes compris~ng a carbohydrate portion; and lig~nd receptors when the analyta comprise~ ~ complemen ar~y ligand, e.g., cell-~uriEace receptor~ ~pecific for proteinaceou~ hormone~.
When the analyte i8 a nucleic acid, the binding 2û moiety can be ssDNA, RNA, or any other n~tural or ~ynthesized ~ingle stranded nucleic acid. Alt~rna-tively the binding moiety could be a non-nucleic: acid molecule which recogllize~ a specific nucleotide sequence, ~uch as an antibody or specific DNA binding protain.
Method~ for the production of antibodie~ or : monoclonal antibodies to be u~ad in the sub~ect invention are known in the literature. 5ee, e.g.~
U.S. Patent No. 4,574,116 and the xeferences cited therein, whose di~closures are herein incorporated by reference. Alternatively, monoclonal antibodie~ or binding fragment3 can be purcha~ed commercially.
. When the binding moiety i8 a nucleic acid molecule, the moiety will usually comprisa at least 12 nuclQotides, more u~ually at least 14 nucleotide~, and pxeferably at least about 18 nucleotides. The size of the binding moiety will vary with the nature oP the analyte, the ~mount of analyte in the sample, and the 2 ~
condition~ employ~d in th~ dstection prvce~. The nucleic acid ~equence~ for u~e in ~ binding moiety can be provided by i~olation from ~ natural Rouxce, synthe~is, or other means known in th~ art.
A sp~cific binding inter~ction must occur between the analyt~ and the binding moiety in order for the assay to be abls to diatingui~3h analyte from othar component~ in the ~ampl~. By ~pecif~c binding interaction i3 meant that the assay can detect ths presence of analyte in tha pre~ence o:E oth~r components normally present in the s~nple. Typically, the binding moiaty for the analyte binds to the analyte only but not to the other component3 of the pl~.
The a~ ay method of the invention can be used to det~ct any analyte that i8 ~uffici2ntly large or h~ multivalent ~ntiyonic ~ite~ to caus~ a meaRurable difference in complementation when bound to the ED-Ab complex ~relativ~ to co~plementation activity in the ab~ence of ~nalyte). Genarally, th~
analyte will be a polyp~ptide, proteln, vlrus particle, polys~ccharide, nucleic acid, lipid, or combination thereof, such as a glycoprotein or lipoprots~n.
Th~ molacular weight of the analyte will usually be at least about lO,OQ0, more u~ually at lea~t about 20,000. Polypeptide~ of intere~t will generally be from about 10,00~ to about 3 x 106 molecular w~ight, more u~ually from about 20,000 to 2 x 106 molecular weight~ Where the analyte i8 a nucleic acid molecule, the molecule will generally range from about 12 nucleotidas to about 2 x 106 nucleotides. Tho nucleic acid sample can involve DN~, which can be chromosomal or extrachromosomal; e.g., plasmids, virusas, synthetic con~tructs, or ~he like;
or RNA, such as messeng2r RNA, tran~fer RN.A, ribosomal RNA, viruses, or the liko. The nucleic acid equences 3 '~ 7 can invol~e ~tructural gene~ on tranxl~ted region~, regulatory regions, intron~, exona, and the like.
The protocol for the a~a~ can be varied widely, depending upon the ~ystem belng employed, the sensitivity of th~ a~say, the ~peed with which ~he a8~ay i8 to be carri~d out, the nature of the analyte, and the like. EA, t~e ED complex, unlabelled antibody (if a multivalent analyt~ i~ being detailed), and 8amplo are combined togethqr under appropriate condition~ of stringeney to allow for binding. The reagents can be combined concomitantly or added sequentially. Where the order i~ sequential, the reaction mixture of ~ampla, ED complex, and buffer i~
preferably incubated for about 5 to 25 minutes, usu~lly about 10 minutes, before the addition of EA, unlabelled ant~body, and enzyma ~ubstrat~.
~ he ~ample can be sub~ected to prior treatment or c~n be u~ed without prior treatment. In the ~ituation in which the analyte in the ~ample i~
capable of binding with the binding moietie~, no prior sample prep~ration i8 generally nec4ssary. In the ~ituat~on in which the analyte in the sample i8 not immediately capable of binding wlth the binding moietiss, prior ~ample preparation will be nece~ary.
For example, whera the analyta i~ double-stranded nucleic acid and the bindin~ moieti~s are complementary nucleic acid strand~, it will be nece~sary to treat the 8ample to denature the double-~tranded molecules before mixing with the ~D complex.
Denaturation can be achieved mo~t readily by sub~ectin~ the sample to high temperature, generally from about 90C to about 100C for about 3 to about 15 minutes. Other means for denaturation can be utilized ~uch a~ treating the sample with alkaline solution~ or concentrated solutions of formamide or through use of other procedure3 known in the art.
The a~ay medium i~ preferably buffered at a pH in the range of about 6 to 9 when u~ing prote:in-1~
h O ~ ~3 3 ~ ~
aceou~ binding moietie~, using a convenient buffex 3uch a3 pho~phate, Tri~, or the like. The ~ignificant factor in xelecting an appropxi~t~ buffer iu that the buffer not inhibit the ~-galactosid~e ~nzyme reaction, complementation of ~A and ED to form active p-galacto~ida~e, or binding of the bindlng moiety to the ~nalyte. Selection of buffer for u~e in diagno~ic a~says, such a~ the pre~ent a~3ay, i~
convention~l. Buffer~ used previously in compl~ment-ation as~ay~ can be used $n the method of the present inventionO
The a~ay can be carried out at any ~ultable temperature which doe~ not inhibit the de~ired reactiun~/ generally at l~a8t at room temperature, which is typirally at least about 20~C/ but prefer~b1y at an eleva~ed temperature below about 40C. The a~ays are generally and preferably parfonmed at atmospheric pressure.
The time~ requ~red for the completion of the desired reaction~ vary depending on the particulars of the assay~. In the situation~, for example, in which the binding ~o$oty 18 a nucle$c acid, the tlme required for hybridization or binding depends on the concentration and ~equenc~ complexity of ths nucleic acid probe, a~ wsll a~ on the assay temper~ture, 801vent, and aalt conc0ntration~. Generally, hybridization i~ carrled out at a temperature of about 20C to about 50C in about O.lS ~ sodium ~hloride and O.015 M sodium citrate for ~ p~riod of about 1/2 hr.
to about lB hr. to allow formation of hybrids.
The technique~ for the hybridization of DNA
are disclo~ed in many references, including Walker and Gaa~tra (eds.) Techniques in Molecular Biology (1983) MacMillan Publi~hing Compsny, New York, pp 113-135 and 273-283; Maniatis et al., (ed~) Molecular Clonin~
( lsa2 ) Cold Spring Harbor Laboratory, pp 309; E.
Southern, J. Mol. Biol. (1975) 98:503; Botchan et al., Cell (1976) 9:269S Jnffrey et al., Cell ~1977) _s 429 . The~e disclosure~ are incorporA~Qd harain by ref~rence .
The amount of sample that i~ u~ed ln con~unctlon with the present invQntiOn depends~ among 5 other things, upon the concentration of the analyte ~
the nature of the 8altlple, and the sen~itivity of the a~ay.
In tha present invention, any ~uitable mean~ can ba u~ed to det~ct and quant~fy the amount of lû active enzyme snd relate the in~Eormation to the datection and determination of the amount of analyte pre~ent in the sampl~. An enzyme ~ub~trate i~
generally and preferably u~ed fox 3uch a purpo~e by providing a measurable product upon re~ction with 15 active p-galactosidase er~ e. The amount of analyte in the sample can than be determinad by comparin~ t:he amount of measurable product to that formed ln tha pre~ence of a known amount of analyte.
The snzyme 3ubstrat~ typically and 20 preerably employed re~ult~ in a ch~nge ir~ the amount of light absorbance (optical density) or emis~ion of : the 8~8ay medium when cleaved by the active enzy~e.
That i~, cleavage of the ~ubstrate re~ults in the appearance or disappearance of a colored or fluorescent product. Preferred enzyme sub~trates include o-nitroph3nyl g~lactosid~ (ONPG) and chlorophenol red-p-galacto~ide (CPRG). ONPG, CPR~, and other comparable enzyme ubstrates are commercially available. ONPG is generally used in a concentration of from about 0.5 to about 2.0 mg/ml.
Other substrates are used in concentrations to provide compa~Able signals to ONPG.
Whexa the ED-Ab complex and EA are combined : in an appropriate assay mediu~ with the enzyme ~ubstrate followed by the subsequent addition of the ~ample, a first reading can be taken to provide a background measurement of enzyma activity. The essential requirement of this background activity i~
3~7 t~at it be di~tingui~hable from activity in the presence of ~he detectabl~ limit of analyte.
After addition of the sample, one or mora additional reading~ can be taken after incubatlon, the interval varying from about 1 minute to about 1 hour, usually about 5 minutes to ~bout 15 minute~, between the readings. While a single ~e~ding ean be taken, it i~ usually de~irable to take more than one rea~ing 80 ~hat common error~ can be cancell~d out.
Preferably, standard aolutions are pr~par~d of known concentratLon~ of analyts to ~erve a~ standard~ for compari on with the sample. In thi~ way, accurate quantitative detenminations can be obt~ined.
The present ~nvention also contemplates a kit containing reagents for carrying out the present inventive ~ethod. Th~ kit co~px.i~es in at least on~
containsr, usually in ~ep~ra e Cont~inerB, a p-g~lac~08 ida~e en2yme donor complex of the invention, an enzyme acceptor, and ~ln preferred embodiment~) unlabelled anti-analyte antibody. Th~
container(~) of enzyme donor complex and enzyme acceptor can additionally con~ain enzyme ~ubstrate, or enzyme sub~trate can be provided ~eparataly.
Alternatively, the kits can be configured 80 that they contain ED attached to a linking group or a precursor of a linkinq group, without a blnding moiety. Upon attachment of p~rticular binding moieties to the ~D
linking elemants of the kit, the desired analyte of interest can be a~sayed. In such cases the linking element~ can be terminated by reactive functional groups for ease o~ attaching to binding moieties supplied by the end u~er.
The followin~ two examples are offered by way of illustration and not by way of limitatlon of the present invention.
1~
E~LE 1 As~av Princi~
In thi~ as~ay the ferxitin ~olecule; having S ~ molecular weight of 480,000, inhibited the ability of an anzyme donor (~D) molecule ~o which an antibody ~pecific fox ferritin WaB ~ound to ~o~plement with ~n enzyme acceptor (~A~ molecule to form actlve p-gal~cto~idase. The amount of inhibition wa~
proportional to the concentration of ferr1tin. In other word~, the hiyher the concentration of ferritin, the lower the activity of p-galacto~idase in the a~say medium, aa mea~ured by color formation resul~ing from react~on of sub~trate with p-galaoto~ida~e.
In the ho~o~eneou~ a~say for the measurement of ~err~tin, ~ampl~8 containing ferritin ware in~ubat~d for 5-10 minutes ~t 37C with an ~D-Ab con~uga ~. The ~D-Ab con~u~at~ WA~ a coval~ntly linksd complex betw~en a~ ~D and ~n antibody u~ing a cros~linking agent. After incubat~on, ~ first reagent (designated Rl) containing ~n antlbody coMplex of antibody to ferritin and sacond~ry antibody wa~
added and incubated ~or 5-10 minute~. ~he Rl reagent al~o contained the ~ub~trste for p-galactosida3e. The ~econd reagent (de~ignated R2~ containing ~A was then add~d to the a~say mixture, ~nd the color signal of the hydrolyzed substrate wa~ spectrophotometrically mea~ured aft~r 4~10 minute3. ~he ferritin amount~ in : sample~ were determined u~ing a calibration curve that was constructed from assay re~ults of a seria~ of samples containing known concentrat~on~ of ferritin.
~aterials Goa~ anti-ferritin antibody purified by affinity column chromatography was purchased from BiosPacific, Inc. ~he hetero-bifunctional crosslinking reagent, sul~o-SMCC, wa~ obtained fro~
Pierce Chemical Company. Chlorophenol red p-galac~o-~ ~ 6 ~ ~ i7 r~
~ide ~CPRG) wa~ purchased from Boehring~r ~annheim~
~nzyme Donor ~nd Enzyme Acceptor (as u~ed in commercial complementation a~$~y8 ) were obt~ined from ~icrogeni~s, Inc. Other chemical~ were purchased from S Si~m~ Chemic~l Co.
Coniuqate Pre~ara~ion The goat anti-ferritin antibody, l ~g in 0.5 ml Buffer A (50 ~M ~odium phosphate, p'.~ 7.4, containing 0.15 ~ N~Cl), wa~ incubated with 50 ~l of 20 mM ~ulfo-SMCC in Buffer A for 15 minute~ at room temp~rature. After the incubation, the mixture waR
loaded on a gel filtration column (PDl0, Pharmacia-LKB) and eluted with Buffer A. ThQ fraction~ wer~
monitored by absorbance ~t 280 nm, and void fraction~
containlng antibody were pooled. One ~g ~D (~D4~ in 0.5 ml Buffer A was thsn added to the pooled fractions and incubated ~or l hour at room temperature. The mixture cont~ining con~ugate w28 concsntrated to 200 ~1 u~ing an ~micon concentrator. Th~ concentrate wa~
loaded on a FP~C SUPEROS~ 12 gel filtrat~on column and elutsd with ~uffer ~ ~50 ~M sodium phosphatQ, pH
.4, 500 m~ N~Cl, 0.05% Tween 20, and 0.05 mM DTT
(dithiothy-reitol)]. Ths fractions were monitor~d b~
absorbance at 280 nm ~nd by complementa~$on activity.
The fr~ction~ containlng antibody &nd complementstion activity were pooled and u~ed ~ the con~ugate.
As~ay_Procedures Procedure l. The as~ay wa~ performed on a COBAS BIO centrifugal analyzer. An a}iquot of 75 ~l of ~ample or c~librator was incubsted with 75 ~l of con~ugate in the Cobas Bio sample cup8 in Buffer C for 10 minute~ at 37C. Buffer C contains 0.1 M MOPS, pH
7.0, 0.4 ~ NaCl, 50 mM EGTA, 3 m~ magne~ium acatat~, 0.05~ Tween 20, 0.05 mM DTT, 3% ethylen~ glycol, and 20 mM ~odium azide. A~ter incubation, the sample cup was placed on the Cobas Bio and 25 ~1 of the mlxture wa~ pipetted and incubated with 155 ~1 of R1 reagent contain~ng anti-farritln antibody, econdary an~ibody, and sub~trate ~CPRG3 in Bufer C, followed by incubation at 37~C for 5 minute~. Thi.rty ~1 of s reagent R2 cont~ining EA in Buf f~r C was then added and incubated for an addition~l 5~10 minute~ at 37C.
The reaction rate was d~termined by measuring absorbance at 570 nm or each ~ample and atandard 801ut ~on .
Procedure 2. Th~ a~say proc:edure 2 wa~
pexformed on ~ Coba~ ~I~ analyzer. The an~lyzer pipatt0d 30 ~1 sample or calibrator fxo~ the 8ample cup and 100 ~1 Rl raagent into cuvette8 and ~ncubated for 14 minutes at 37C. The Rl reagent contained the 15 Ab-~D con~u~ate and the aubst:rate ( CP~G) in buf fer C .
After incubation, 75 ~41 of R2 reagen~ wa~ pip~tted ancl lncub~ted for 3-7 minut~ at 37C. The R~ xeagent contained EA, ~nti-ferritin antibody, and ~econdary ant~body in the bu~Per ~. ~he reaction rat0 wa~
detenmined by mea~urlng absorbance at 550 nm for each ~ample and standasd ~olution.
Results A calibration curve wa~ con~tructed u~ing a series of known ferritin concentrations from 0 to 500 ng~l. The ~alibration curve i~ qhown in Figure 1 (procedure 1) or Figur~ 4 (procedure 2). The calibration curve i8 linear from a high rate (mea~ured in ~AU/min) for zero fsrrit~n concentration to a low rate at 500 ng/ml ferritin. A highly ~ensitive calibration curve f~r the detection of low-end concantration~ of ~erritin i8 shown in Figura 2.
Usins the calibration curve of Figure 2 or Figure 4, d~tection sensltivity o 6 ng/ml ferritin i~ achioved by thi~ homogeneous a3say procedure. The ferritin concentrations in 72 human sera have been correctly I measured by the a~ay of the invention as indicated by ! compari30n ~o a FDA-approvod reference procedur3. The i Q3 ~17 correlat$on curve i8 ~I~OWn in Figure 3. A~ ~hown ln Fi5~ure 5, the procedure 2 ~l~o mea~ureù f~rr~tin in 45 humall ~er~ correctly. This ~aethod provide~ ~ fully autom~ted, f a~t and ~ccurate homogenou~ nunoa~say 5 for ferritin unlike the other methods which require mult ~ ple ~teps and r~quire colaplea~ in~trumentat$on .
Y X.WLY 2 In th~ ~ a~ay the C-Reactiv~ Protein molecul~, having a molecular w~ight of 125, 000, inhibitad the ab~lity of an enzyme donor (~D) molecul~ to whi~h an antibody specific for t::-Reactive Protein wa~ bound to complement with an enzyme acceptor (~A~ molecllla to 15 form active ~-galacto~ida~e. Th~ amoun~ of inhibi~ion wa~ proportional to the concen'cration of C-Reactiv~s Protein. In other words, the hig~h~r th~3 ooncentration of C Re~acti-ve Protein, th~ low~r the ~ctivity o gal~cto~idas~ in thc ~ssay m~dium, as me~sured by 20 color formation re~ulting fro~ reaction of sub~trate with p-galactosida~eO
In the homogeneous a~ay for the mea~urement of C-Reactive Protein, 8ample8 containin~ C-~eactive Protein were incubated for 5-lO min at 37 with an ED-Ab(monoclon~l) con~ugate and another uncon~ugated Ab(monoclon~l). The ED-Ab con~ug~te wa~ ~ covalently ; linked comple~ between an ED and ~n Ab using a cro~
linking agent. ~f~er incubation, a first reagent (de~ignated RI~ containing a secondary anti~ody to mou~e IgG and a substrate for p-galacto3idase was added and incubated for 5-10 m$nutes. The second reagent (designated R2) containin~ EA waa then added to the a~say mixture, and the color slgnal of the hydrolyzed 3ub~trate was ~pectrophotometrically mea~ured after 4-~0 minute~. The C-react$ve protein amounts in 8ample8 were deter~ined u~ing a calibration curve that was con~tructed from assay reqults of a ~8 2 ~ 7 ~
seri~ of ~ample~ contain~ng known concentr~tion~ of C-reactive protein.
Mate Monoclonal ~nti C-reactive protein antibod~a were purchas~d from ~edix 3iochemica. The h~tero-bifunct$on~1 cro~slinking reagent, sulfo SMCC, W~3 obta$ned from Piarce Ch~ic~l C~mpany. Chlorophenol red p-galacto~id~ (CPRG) W88 purcha3ed from Boehringer ~annhei~. Enzym~ ~onor and ~nzyme Acceptor ta~ u~ed in commercial complementation a~3~y~) were obt~ined from Mlcrogenic~, Inc. OthQr chemic~l3 were purcha~ed from Si~ma Chemical Co.
Coniuqate Prepara~ion The monoclonal anti-C-roactiYe protein antibody, 1 mg in 0.5 ml Buffer ~ (50 m~ sodiu~ pho~phate, pH
7.4, containin~ 0.15 M NaCl), w~8 incubated with 50 ~1 of 20 m~ ~ulfo-SMCC in Buffer A for 15 ~ins at room temperstur2. ~fter th~ incubation, the mixtuxs was loaded on a gel f~ltration ~ol~mn (PD10, Phar~aci~-LXB) and ~luted with ~uffer ~. Tho fract~on~ were monitored by ~bsorbanc~ st 280 nm, and void fraction~
containing antibody were pooled. One mg ED (ED4~ in 0.5 ml Buffer A wa~ then added to the pooled fraction~
and incubat~d for 1 hour at room temper~ture. The mixture containing con~ugats wa~ concentrated to 200 ~1 using ~n Amicon concentrator. ~he concentrate wa~ loaded on a FPLC Supero~e 12 gel filtration col~nn and eluted with Buffsr B [50 mM ~odium phoQphate, pH
7.4, 500 m~ NaCl, G.05~ Tw~en 20, and 0.05 mM DTT
~dithiothy-reitol)]. The fractions were monitored by ab30rbance at 280 nm and by complementation activity.
The ~raction~ containing antibody and complementation activity were poolad and used as ths con~ugate.
A~ay Pxocedure~
j 35 The a88ay wa8 parformed on a Coba~ Bio ¦ centrifugal analyz~r. An aliquot of 3ample was diluted 10-fold with ~GTA buffer~ An aliguot of 7 ~
10 . 5 ,~1 of the diluted ~mple wa~ incub~t~d with 129 . S
~1 of con~u~ated and uncon~ugated ~n~-C-r~ctive protein antibody in EGTA buff~r for 10 min ~t 37.
Af~er incubation, the 8~3mpl2 CUp wa~ pl~ced on ~he 5 Cobas 3io ~nd 10 ~1 of the ~ixture was pipetl:ecl and incubated with 175 ~1 of Rl reag~n~ eont~ining ~e~ondary antibody ~nd ~ub~rat~ t CPRG ) in I :GTA
buf fer . Incubation ~t this step wa~ for 10 min .
Thirty ~1 of -reagent R2 containin~ EA in Buf ~g~r C was 10 then addQd ~nd ~ncubat~d for an addi'cional 5-10 min at 37C. The re~ction r~e ~ra~ deterrain~ed by ~e~urirlg absorb~nc~ at 574 nM for e~ch sampla and ~tarldard 801ult~0n.
E:GTA buffer cont~ins 150 mM pota~ phosphate, 100 mPI ~odiu~ pho~phat~, 3 m~ m~gn~asium ac~at~te, 0.059 Tween 20, 0.05 ~M DTT, 1.2~ ethyl0n~ glycol~ and 20 m~
~odium a~ide, at ~ final pH 7Ø
Re~ult~
A calibrntion curve was con~tructed using a 20 8er~e~ o~ ~nown C-reactiv~-protain concentr~tions from O to 120 ~g/ml. The calibration curve i8 ~hown ~n Flg. 6. Th~ calibration curve i~ a non-linsar curve from ~ high r~te (~ea~ured in mAu/min) ~or ~ero C-reactive prok~ln to a low xate ~t 120 ~g/ml C-re~ct~ve ~5 proteinO Th~ u~e of ~ no~-line~r curve ~llows for low end sen~itivity while ~aint~ining a bro~d a83ay r~nga.
Using the calibration cur~e of Fig 6, a detection ~en~itivity of l~g/ml C reactiYe protein can b~
achieved. The C-reactive protein concentrations in 11 human 3era have been correctly mea~ured by the assay of the invention as indicated by comparison to a FDA-approved reference procedure. The correlation curve i~ shown in FigNre 7.
It i~ evid~nt fro~ the above reault~ tha~ the sub~ect ~e~hod pro~ides for an accurat~, sen~itive, and rapid technique for detecting levels of high-molecular-weight analytes, particularly in a complex 2 ~ 7 ~i ~ixt~re. The ob~erved en~yme actlvity i~ inv~r~ely proportioll~l to the amount of analyte ln the 8alllple.
All publication~ and p~tent applicstionE cited in thi~ speci~icatlon are herein ~ncorporated ~y 5 reference a~ if each individual public~t:Lon or patent application were ~pecifically and individually indicated to be incorporated by refer0nce.
Although tlle foregoinq invention ha~ b~en de~crib~3d in ~ome detAil by way of illustration ~nd 10 exainple for purpo~e~ of clarity of urlder~tanding, i~
will be readily appar~nt to tho~e of ordillary ~k~ 11 in the art in light of the teaching~ of this inven'cion that certain change~ and modific~tions can be made ther2to without departing from the ~pirit or ~cope of 15 the appended claim3.
~1
coupled to an analyte-specific bindlng molecula, wherein said ED complex retain~ mes~urable complementation activity ~uch that active ~-~alactosida~e i~ formed in the presence of EA;
(b) contacting th~ ED complex and E~ (andan exc~s~ unlabelled binding moiety in preferred embodiments u ing ~ultivalen~ analyte~) in ~he pre~ence of a aample 3uspected of containing a high~
molecular-weight analyte that react~ ~peciically wi~h the analyte-~pecific binding molecule, and (c) relating the pres~nce of the analyte in the sample to the fonmation of active ~-galacto~idase enzyme.
lS The present invention ~ predicated, at least in part, on the principle that the ~D/bindiny-moiety complex and EA for~ actLve enz~ne at a differont rate than does the combination of E~ with the ternary complex of ~D/binding-moiety/high-molecular-weight analyte. When a ~m 11 amount of the analyts i~ pre~ent, only a part o~ ED-Ab con~uga~e can bind to the analyte and hence more of ED-Ab con~ugate i~ available for complem~ntation with EA. To the contrary, when large amount~ of analyte are pre~ent, le~s ED-~b coniugate i~ available for complementstion with EA, and hencs, les~ enzyme activity i3 ob~er~ed.
In both case3, multivalent antigenic determinants allow the s~multaneous complexation of ED-binding moiety con~ugate and unla~elled anti-analyte antibody binding at multiple ~ite~. Unlabelled antibody occupie3 the unbound 8ite8 and makes enzyme complementation inhibition more effective. The ~ize I of the ternary complex, consi~ting of ED-~h con~ugate, analyte, snd unlabelled antibody become~
very large per ED molecule, and hence les3 complementation i3 observed. The quanti~y of the analyte, therefore, i~ in~ersely proportional to h~3 ~
enzyms activity, which c~n be measured in any ~uitable manner, as through the u~e of an enzyme ~ub~ra~e.
The present inventive method therefore further contemp}ates the ED-~b complex t EA~ unlabelled ant~body, and sample being contacted with an enz~me sub~trate which provides or a mea~ur~ble product upon reaction with complementad p-galactosida~e such that the amount of analyte in the ~ample can be de~enmined by comparing the amount of measurable product to that formed in th~ presence of a kn~wn amount of analyte.
The pre~ent invention al80 contemplates kit~ for use in carrying out the method of the pre~ent invention as well as individual reagent~ used in the method of the invention.
The ability of ths ED-~b complex of the present invention to complement with EA in the absence of analyte differ~ markedly from ~he p-galactosidase complementation phenomenon observed in a number of prev$ou~1y described assays employing ED and E~
con~ugatea. For example, in the a~saya describ~d in US Patent 4,708,929, ED con~ugated to a small analyte complement~ with EA in the absence of anti-analyte antibodie3. ~hen the anti-analyte antibodiex are pre~ent in the assay mixture, complementation activity drops ~ignificantly when compared to initial activity in the ~bsence of antibodies. In the pxe~ence of analyte present in the unknown sample, a competition occurs between the ~D-labelled analyte and the analyte present in the sample, for a limited amount of binding sites. Thux, pre~ence of more analyte ties up more antibody and hence, allows corre4pondingly more ED-analyte to complement with EA. T}lus, complemented enzyme ~ctivity i8 directly proportional to the amount of analyte in the sample. Thi~ competitive assay method works well only with ~mall molecules.
In contra~t, the present invention i8 a non-competitive l~munoas~ay. An ED-binding moiety con~ugate is employed as compared to ED-hapten 3 ~ ~
con~ugate in the previously described method.
Further, the pre~eng method allow~ a direct assay where the analyte to be measured i~ u~ed to inhibit the rate of enzyme complementation. In addition, the present as~ay principle works well with high molecular weight analyte~ preferably having mult:L~alent antig&nic determinant~.
The difference in the prior teaching and the pre ent re8ult8 i~ believed to result from ~he large size of the analyte/ED-Ab~unlabelled Ab complex relati~e to the bLnding moietie~. By using the present a ~ay only when the analytes are very large, it i~ 8till po~sible to maint~in a difference in complementation rate~. Such difference~ would have been undetectable on ~he ~cal* of complementat$on rates known previously for prior as~ays in which ~mall analytes were att~ched to ED molecule~ and allowed to react with antibodies to reduce the complementation rate.
The ~ and EA componant~ of the pre~en~
invention are partia} sequences of p-galacto~idase.
For the purpos~ of the sub~ect invention, the shorter portion of the p-galactosidase molecule i~ referred to a~ the enzyme donor (~D), and the longer portion i~
the enzyme acceptor ~EA). The enzyme acceptor and enzyme donor are character$zed by forming an active enzyme complex when brought together. The preparation of ~-galacto~idase enzyme donor~ and acceptor~ is described in U.S. Patent No. 4,708,929, which disclo~ure i~ incorporated herein by reference.
Any of the previously described methods for attaching proteins to other molecules can be u~ed to attach the ED component to the binding mo~ety.
Especially preferred i8 the u3a of bifunctionsl organic linking group~ to attach proteinaceou~ binding moietie~ to the ED fragm~nt, which it~el~ i8 proteinaceou~. Example-q of such linking groups, which are well known in protein chemi~try, include 7~
dialdehyd4~, ~uch a~ glut~ral~ehyde, and diamlne~, such aR 1, 6-dlaminohexane as described in the ~pecif ic literature (e.g., J. In~nunoa~say, 1983, 4~209).
The binding moieties are components capable of 5 binding to t}le particular analyl:e of intere~t. The binding between the bind$n~ moietie~ and the an~lyte i~ preferably non-coval~nt. 5uitable binding moietie~
preferably have higher affin~ty and ~pecificity for the analyte than for the other components in a ~ample 10 being analyzed. Suitabla binding moieties can be of a variety of molecular categorie~ includlng antibodie~
specific for a portion of the analyte ~prefexably, monoclonal antibodie~ ); binding protein~ that naturally blnd to the analyte , e . S~ ., lectins iEor 15 analytes compris~ng a carbohydrate portion; and lig~nd receptors when the analyta comprise~ ~ complemen ar~y ligand, e.g., cell-~uriEace receptor~ ~pecific for proteinaceou~ hormone~.
When the analyte i8 a nucleic acid, the binding 2û moiety can be ssDNA, RNA, or any other n~tural or ~ynthesized ~ingle stranded nucleic acid. Alt~rna-tively the binding moiety could be a non-nucleic: acid molecule which recogllize~ a specific nucleotide sequence, ~uch as an antibody or specific DNA binding protain.
Method~ for the production of antibodie~ or : monoclonal antibodies to be u~ad in the sub~ect invention are known in the literature. 5ee, e.g.~
U.S. Patent No. 4,574,116 and the xeferences cited therein, whose di~closures are herein incorporated by reference. Alternatively, monoclonal antibodie~ or binding fragment3 can be purcha~ed commercially.
. When the binding moiety i8 a nucleic acid molecule, the moiety will usually comprisa at least 12 nuclQotides, more u~ually at least 14 nucleotide~, and pxeferably at least about 18 nucleotides. The size of the binding moiety will vary with the nature oP the analyte, the ~mount of analyte in the sample, and the 2 ~
condition~ employ~d in th~ dstection prvce~. The nucleic acid ~equence~ for u~e in ~ binding moiety can be provided by i~olation from ~ natural Rouxce, synthe~is, or other means known in th~ art.
A sp~cific binding inter~ction must occur between the analyt~ and the binding moiety in order for the assay to be abls to diatingui~3h analyte from othar component~ in the ~ampl~. By ~pecif~c binding interaction i3 meant that the assay can detect ths presence of analyte in tha pre~ence o:E oth~r components normally present in the s~nple. Typically, the binding moiaty for the analyte binds to the analyte only but not to the other component3 of the pl~.
The a~ ay method of the invention can be used to det~ct any analyte that i8 ~uffici2ntly large or h~ multivalent ~ntiyonic ~ite~ to caus~ a meaRurable difference in complementation when bound to the ED-Ab complex ~relativ~ to co~plementation activity in the ab~ence of ~nalyte). Genarally, th~
analyte will be a polyp~ptide, proteln, vlrus particle, polys~ccharide, nucleic acid, lipid, or combination thereof, such as a glycoprotein or lipoprots~n.
Th~ molacular weight of the analyte will usually be at least about lO,OQ0, more u~ually at lea~t about 20,000. Polypeptide~ of intere~t will generally be from about 10,00~ to about 3 x 106 molecular w~ight, more u~ually from about 20,000 to 2 x 106 molecular weight~ Where the analyte i8 a nucleic acid molecule, the molecule will generally range from about 12 nucleotidas to about 2 x 106 nucleotides. Tho nucleic acid sample can involve DN~, which can be chromosomal or extrachromosomal; e.g., plasmids, virusas, synthetic con~tructs, or ~he like;
or RNA, such as messeng2r RNA, tran~fer RN.A, ribosomal RNA, viruses, or the liko. The nucleic acid equences 3 '~ 7 can invol~e ~tructural gene~ on tranxl~ted region~, regulatory regions, intron~, exona, and the like.
The protocol for the a~a~ can be varied widely, depending upon the ~ystem belng employed, the sensitivity of th~ a~say, the ~peed with which ~he a8~ay i8 to be carri~d out, the nature of the analyte, and the like. EA, t~e ED complex, unlabelled antibody (if a multivalent analyt~ i~ being detailed), and 8amplo are combined togethqr under appropriate condition~ of stringeney to allow for binding. The reagents can be combined concomitantly or added sequentially. Where the order i~ sequential, the reaction mixture of ~ampla, ED complex, and buffer i~
preferably incubated for about 5 to 25 minutes, usu~lly about 10 minutes, before the addition of EA, unlabelled ant~body, and enzyma ~ubstrat~.
~ he ~ample can be sub~ected to prior treatment or c~n be u~ed without prior treatment. In the ~ituation in which the analyte in the ~ample i~
capable of binding with the binding moietie~, no prior sample prep~ration i8 generally nec4ssary. In the ~ituat~on in which the analyte in the sample i8 not immediately capable of binding wlth the binding moietiss, prior ~ample preparation will be nece~ary.
For example, whera the analyta i~ double-stranded nucleic acid and the bindin~ moieti~s are complementary nucleic acid strand~, it will be nece~sary to treat the 8ample to denature the double-~tranded molecules before mixing with the ~D complex.
Denaturation can be achieved mo~t readily by sub~ectin~ the sample to high temperature, generally from about 90C to about 100C for about 3 to about 15 minutes. Other means for denaturation can be utilized ~uch a~ treating the sample with alkaline solution~ or concentrated solutions of formamide or through use of other procedure3 known in the art.
The a~ay medium i~ preferably buffered at a pH in the range of about 6 to 9 when u~ing prote:in-1~
h O ~ ~3 3 ~ ~
aceou~ binding moietie~, using a convenient buffex 3uch a3 pho~phate, Tri~, or the like. The ~ignificant factor in xelecting an appropxi~t~ buffer iu that the buffer not inhibit the ~-galactosid~e ~nzyme reaction, complementation of ~A and ED to form active p-galacto~ida~e, or binding of the bindlng moiety to the ~nalyte. Selection of buffer for u~e in diagno~ic a~says, such a~ the pre~ent a~3ay, i~
convention~l. Buffer~ used previously in compl~ment-ation as~ay~ can be used $n the method of the present inventionO
The a~ay can be carried out at any ~ultable temperature which doe~ not inhibit the de~ired reactiun~/ generally at l~a8t at room temperature, which is typirally at least about 20~C/ but prefer~b1y at an eleva~ed temperature below about 40C. The a~ays are generally and preferably parfonmed at atmospheric pressure.
The time~ requ~red for the completion of the desired reaction~ vary depending on the particulars of the assay~. In the situation~, for example, in which the binding ~o$oty 18 a nucle$c acid, the tlme required for hybridization or binding depends on the concentration and ~equenc~ complexity of ths nucleic acid probe, a~ wsll a~ on the assay temper~ture, 801vent, and aalt conc0ntration~. Generally, hybridization i~ carrled out at a temperature of about 20C to about 50C in about O.lS ~ sodium ~hloride and O.015 M sodium citrate for ~ p~riod of about 1/2 hr.
to about lB hr. to allow formation of hybrids.
The technique~ for the hybridization of DNA
are disclo~ed in many references, including Walker and Gaa~tra (eds.) Techniques in Molecular Biology (1983) MacMillan Publi~hing Compsny, New York, pp 113-135 and 273-283; Maniatis et al., (ed~) Molecular Clonin~
( lsa2 ) Cold Spring Harbor Laboratory, pp 309; E.
Southern, J. Mol. Biol. (1975) 98:503; Botchan et al., Cell (1976) 9:269S Jnffrey et al., Cell ~1977) _s 429 . The~e disclosure~ are incorporA~Qd harain by ref~rence .
The amount of sample that i~ u~ed ln con~unctlon with the present invQntiOn depends~ among 5 other things, upon the concentration of the analyte ~
the nature of the 8altlple, and the sen~itivity of the a~ay.
In tha present invention, any ~uitable mean~ can ba u~ed to det~ct and quant~fy the amount of lû active enzyme snd relate the in~Eormation to the datection and determination of the amount of analyte pre~ent in the sampl~. An enzyme ~ub~trate i~
generally and preferably u~ed fox 3uch a purpo~e by providing a measurable product upon re~ction with 15 active p-galactosidase er~ e. The amount of analyte in the sample can than be determinad by comparin~ t:he amount of measurable product to that formed ln tha pre~ence of a known amount of analyte.
The snzyme 3ubstrat~ typically and 20 preerably employed re~ult~ in a ch~nge ir~ the amount of light absorbance (optical density) or emis~ion of : the 8~8ay medium when cleaved by the active enzy~e.
That i~, cleavage of the ~ubstrate re~ults in the appearance or disappearance of a colored or fluorescent product. Preferred enzyme sub~trates include o-nitroph3nyl g~lactosid~ (ONPG) and chlorophenol red-p-galacto~ide (CPRG). ONPG, CPR~, and other comparable enzyme ubstrates are commercially available. ONPG is generally used in a concentration of from about 0.5 to about 2.0 mg/ml.
Other substrates are used in concentrations to provide compa~Able signals to ONPG.
Whexa the ED-Ab complex and EA are combined : in an appropriate assay mediu~ with the enzyme ~ubstrate followed by the subsequent addition of the ~ample, a first reading can be taken to provide a background measurement of enzyma activity. The essential requirement of this background activity i~
3~7 t~at it be di~tingui~hable from activity in the presence of ~he detectabl~ limit of analyte.
After addition of the sample, one or mora additional reading~ can be taken after incubatlon, the interval varying from about 1 minute to about 1 hour, usually about 5 minutes to ~bout 15 minute~, between the readings. While a single ~e~ding ean be taken, it i~ usually de~irable to take more than one rea~ing 80 ~hat common error~ can be cancell~d out.
Preferably, standard aolutions are pr~par~d of known concentratLon~ of analyts to ~erve a~ standard~ for compari on with the sample. In thi~ way, accurate quantitative detenminations can be obt~ined.
The present ~nvention also contemplates a kit containing reagents for carrying out the present inventive ~ethod. Th~ kit co~px.i~es in at least on~
containsr, usually in ~ep~ra e Cont~inerB, a p-g~lac~08 ida~e en2yme donor complex of the invention, an enzyme acceptor, and ~ln preferred embodiment~) unlabelled anti-analyte antibody. Th~
container(~) of enzyme donor complex and enzyme acceptor can additionally con~ain enzyme ~ubstrate, or enzyme sub~trate can be provided ~eparataly.
Alternatively, the kits can be configured 80 that they contain ED attached to a linking group or a precursor of a linkinq group, without a blnding moiety. Upon attachment of p~rticular binding moieties to the ~D
linking elemants of the kit, the desired analyte of interest can be a~sayed. In such cases the linking element~ can be terminated by reactive functional groups for ease o~ attaching to binding moieties supplied by the end u~er.
The followin~ two examples are offered by way of illustration and not by way of limitatlon of the present invention.
1~
E~LE 1 As~av Princi~
In thi~ as~ay the ferxitin ~olecule; having S ~ molecular weight of 480,000, inhibited the ability of an anzyme donor (~D) molecule ~o which an antibody ~pecific fox ferritin WaB ~ound to ~o~plement with ~n enzyme acceptor (~A~ molecule to form actlve p-gal~cto~idase. The amount of inhibition wa~
proportional to the concentration of ferr1tin. In other word~, the hiyher the concentration of ferritin, the lower the activity of p-galacto~idase in the a~say medium, aa mea~ured by color formation resul~ing from react~on of sub~trate with p-galaoto~ida~e.
In the ho~o~eneou~ a~say for the measurement of ~err~tin, ~ampl~8 containing ferritin ware in~ubat~d for 5-10 minutes ~t 37C with an ~D-Ab con~uga ~. The ~D-Ab con~u~at~ WA~ a coval~ntly linksd complex betw~en a~ ~D and ~n antibody u~ing a cros~linking agent. After incubat~on, ~ first reagent (designated Rl) containing ~n antlbody coMplex of antibody to ferritin and sacond~ry antibody wa~
added and incubated ~or 5-10 minute~. ~he Rl reagent al~o contained the ~ub~trste for p-galactosida3e. The ~econd reagent (de~ignated R2~ containing ~A was then add~d to the a~say mixture, ~nd the color signal of the hydrolyzed substrate wa~ spectrophotometrically mea~ured aft~r 4~10 minute3. ~he ferritin amount~ in : sample~ were determined u~ing a calibration curve that was constructed from assay re~ults of a seria~ of samples containing known concentrat~on~ of ferritin.
~aterials Goa~ anti-ferritin antibody purified by affinity column chromatography was purchased from BiosPacific, Inc. ~he hetero-bifunctional crosslinking reagent, sul~o-SMCC, wa~ obtained fro~
Pierce Chemical Company. Chlorophenol red p-galac~o-~ ~ 6 ~ ~ i7 r~
~ide ~CPRG) wa~ purchased from Boehring~r ~annheim~
~nzyme Donor ~nd Enzyme Acceptor (as u~ed in commercial complementation a~$~y8 ) were obt~ined from ~icrogeni~s, Inc. Other chemical~ were purchased from S Si~m~ Chemic~l Co.
Coniuqate Pre~ara~ion The goat anti-ferritin antibody, l ~g in 0.5 ml Buffer A (50 ~M ~odium phosphate, p'.~ 7.4, containing 0.15 ~ N~Cl), wa~ incubated with 50 ~l of 20 mM ~ulfo-SMCC in Buffer A for 15 minute~ at room temp~rature. After the incubation, the mixture waR
loaded on a gel filtration column (PDl0, Pharmacia-LKB) and eluted with Buffer A. ThQ fraction~ wer~
monitored by absorbance ~t 280 nm, and void fraction~
containlng antibody were pooled. One ~g ~D (~D4~ in 0.5 ml Buffer A was thsn added to the pooled fractions and incubated ~or l hour at room temperature. The mixture cont~ining con~ugate w28 concsntrated to 200 ~1 u~ing an ~micon concentrator. Th~ concentrate wa~
loaded on a FP~C SUPEROS~ 12 gel filtrat~on column and elutsd with ~uffer ~ ~50 ~M sodium phosphatQ, pH
.4, 500 m~ N~Cl, 0.05% Tween 20, and 0.05 mM DTT
(dithiothy-reitol)]. Ths fractions were monitor~d b~
absorbance at 280 nm ~nd by complementa~$on activity.
The fr~ction~ containlng antibody &nd complementstion activity were pooled and u~ed ~ the con~ugate.
As~ay_Procedures Procedure l. The as~ay wa~ performed on a COBAS BIO centrifugal analyzer. An a}iquot of 75 ~l of ~ample or c~librator was incubsted with 75 ~l of con~ugate in the Cobas Bio sample cup8 in Buffer C for 10 minute~ at 37C. Buffer C contains 0.1 M MOPS, pH
7.0, 0.4 ~ NaCl, 50 mM EGTA, 3 m~ magne~ium acatat~, 0.05~ Tween 20, 0.05 mM DTT, 3% ethylen~ glycol, and 20 mM ~odium azide. A~ter incubation, the sample cup was placed on the Cobas Bio and 25 ~1 of the mlxture wa~ pipetted and incubated with 155 ~1 of R1 reagent contain~ng anti-farritln antibody, econdary an~ibody, and sub~trate ~CPRG3 in Bufer C, followed by incubation at 37~C for 5 minute~. Thi.rty ~1 of s reagent R2 cont~ining EA in Buf f~r C was then added and incubated for an addition~l 5~10 minute~ at 37C.
The reaction rate was d~termined by measuring absorbance at 570 nm or each ~ample and atandard 801ut ~on .
Procedure 2. Th~ a~say proc:edure 2 wa~
pexformed on ~ Coba~ ~I~ analyzer. The an~lyzer pipatt0d 30 ~1 sample or calibrator fxo~ the 8ample cup and 100 ~1 Rl raagent into cuvette8 and ~ncubated for 14 minutes at 37C. The Rl reagent contained the 15 Ab-~D con~u~ate and the aubst:rate ( CP~G) in buf fer C .
After incubation, 75 ~41 of R2 reagen~ wa~ pip~tted ancl lncub~ted for 3-7 minut~ at 37C. The R~ xeagent contained EA, ~nti-ferritin antibody, and ~econdary ant~body in the bu~Per ~. ~he reaction rat0 wa~
detenmined by mea~urlng absorbance at 550 nm for each ~ample and standasd ~olution.
Results A calibration curve wa~ con~tructed u~ing a series of known ferritin concentrations from 0 to 500 ng~l. The ~alibration curve i~ qhown in Figure 1 (procedure 1) or Figur~ 4 (procedure 2). The calibration curve i8 linear from a high rate (mea~ured in ~AU/min) for zero fsrrit~n concentration to a low rate at 500 ng/ml ferritin. A highly ~ensitive calibration curve f~r the detection of low-end concantration~ of ~erritin i8 shown in Figura 2.
Usins the calibration curve of Figure 2 or Figure 4, d~tection sensltivity o 6 ng/ml ferritin i~ achioved by thi~ homogeneous a3say procedure. The ferritin concentrations in 72 human sera have been correctly I measured by the a~ay of the invention as indicated by ! compari30n ~o a FDA-approvod reference procedur3. The i Q3 ~17 correlat$on curve i8 ~I~OWn in Figure 3. A~ ~hown ln Fi5~ure 5, the procedure 2 ~l~o mea~ureù f~rr~tin in 45 humall ~er~ correctly. This ~aethod provide~ ~ fully autom~ted, f a~t and ~ccurate homogenou~ nunoa~say 5 for ferritin unlike the other methods which require mult ~ ple ~teps and r~quire colaplea~ in~trumentat$on .
Y X.WLY 2 In th~ ~ a~ay the C-Reactiv~ Protein molecul~, having a molecular w~ight of 125, 000, inhibitad the ab~lity of an enzyme donor (~D) molecul~ to whi~h an antibody specific for t::-Reactive Protein wa~ bound to complement with an enzyme acceptor (~A~ molecllla to 15 form active ~-galacto~ida~e. Th~ amoun~ of inhibi~ion wa~ proportional to the concen'cration of C-Reactiv~s Protein. In other words, the hig~h~r th~3 ooncentration of C Re~acti-ve Protein, th~ low~r the ~ctivity o gal~cto~idas~ in thc ~ssay m~dium, as me~sured by 20 color formation re~ulting fro~ reaction of sub~trate with p-galactosida~eO
In the homogeneous a~ay for the mea~urement of C-Reactive Protein, 8ample8 containin~ C-~eactive Protein were incubated for 5-lO min at 37 with an ED-Ab(monoclon~l) con~ugate and another uncon~ugated Ab(monoclon~l). The ED-Ab con~ug~te wa~ ~ covalently ; linked comple~ between an ED and ~n Ab using a cro~
linking agent. ~f~er incubation, a first reagent (de~ignated RI~ containing a secondary anti~ody to mou~e IgG and a substrate for p-galacto3idase was added and incubated for 5-10 m$nutes. The second reagent (designated R2) containin~ EA waa then added to the a~say mixture, and the color slgnal of the hydrolyzed 3ub~trate was ~pectrophotometrically mea~ured after 4-~0 minute~. The C-react$ve protein amounts in 8ample8 were deter~ined u~ing a calibration curve that was con~tructed from assay reqults of a ~8 2 ~ 7 ~
seri~ of ~ample~ contain~ng known concentr~tion~ of C-reactive protein.
Mate Monoclonal ~nti C-reactive protein antibod~a were purchas~d from ~edix 3iochemica. The h~tero-bifunct$on~1 cro~slinking reagent, sulfo SMCC, W~3 obta$ned from Piarce Ch~ic~l C~mpany. Chlorophenol red p-galacto~id~ (CPRG) W88 purcha3ed from Boehringer ~annhei~. Enzym~ ~onor and ~nzyme Acceptor ta~ u~ed in commercial complementation a~3~y~) were obt~ined from Mlcrogenic~, Inc. OthQr chemic~l3 were purcha~ed from Si~ma Chemical Co.
Coniuqate Prepara~ion The monoclonal anti-C-roactiYe protein antibody, 1 mg in 0.5 ml Buffer ~ (50 m~ sodiu~ pho~phate, pH
7.4, containin~ 0.15 M NaCl), w~8 incubated with 50 ~1 of 20 m~ ~ulfo-SMCC in Buffer A for 15 ~ins at room temperstur2. ~fter th~ incubation, the mixtuxs was loaded on a gel f~ltration ~ol~mn (PD10, Phar~aci~-LXB) and ~luted with ~uffer ~. Tho fract~on~ were monitored by ~bsorbanc~ st 280 nm, and void fraction~
containing antibody were pooled. One mg ED (ED4~ in 0.5 ml Buffer A wa~ then added to the pooled fraction~
and incubat~d for 1 hour at room temper~ture. The mixture containing con~ugats wa~ concentrated to 200 ~1 using ~n Amicon concentrator. ~he concentrate wa~ loaded on a FPLC Supero~e 12 gel filtration col~nn and eluted with Buffsr B [50 mM ~odium phoQphate, pH
7.4, 500 m~ NaCl, G.05~ Tw~en 20, and 0.05 mM DTT
~dithiothy-reitol)]. The fractions were monitored by ab30rbance at 280 nm and by complementation activity.
The ~raction~ containing antibody and complementation activity were poolad and used as ths con~ugate.
A~ay Pxocedure~
j 35 The a88ay wa8 parformed on a Coba~ Bio ¦ centrifugal analyz~r. An aliquot of 3ample was diluted 10-fold with ~GTA buffer~ An aliguot of 7 ~
10 . 5 ,~1 of the diluted ~mple wa~ incub~t~d with 129 . S
~1 of con~u~ated and uncon~ugated ~n~-C-r~ctive protein antibody in EGTA buff~r for 10 min ~t 37.
Af~er incubation, the 8~3mpl2 CUp wa~ pl~ced on ~he 5 Cobas 3io ~nd 10 ~1 of the ~ixture was pipetl:ecl and incubated with 175 ~1 of Rl reag~n~ eont~ining ~e~ondary antibody ~nd ~ub~rat~ t CPRG ) in I :GTA
buf fer . Incubation ~t this step wa~ for 10 min .
Thirty ~1 of -reagent R2 containin~ EA in Buf ~g~r C was 10 then addQd ~nd ~ncubat~d for an addi'cional 5-10 min at 37C. The re~ction r~e ~ra~ deterrain~ed by ~e~urirlg absorb~nc~ at 574 nM for e~ch sampla and ~tarldard 801ult~0n.
E:GTA buffer cont~ins 150 mM pota~ phosphate, 100 mPI ~odiu~ pho~phat~, 3 m~ m~gn~asium ac~at~te, 0.059 Tween 20, 0.05 ~M DTT, 1.2~ ethyl0n~ glycol~ and 20 m~
~odium a~ide, at ~ final pH 7Ø
Re~ult~
A calibrntion curve was con~tructed using a 20 8er~e~ o~ ~nown C-reactiv~-protain concentr~tions from O to 120 ~g/ml. The calibration curve i8 ~hown ~n Flg. 6. Th~ calibration curve i~ a non-linsar curve from ~ high r~te (~ea~ured in mAu/min) ~or ~ero C-reactive prok~ln to a low xate ~t 120 ~g/ml C-re~ct~ve ~5 proteinO Th~ u~e of ~ no~-line~r curve ~llows for low end sen~itivity while ~aint~ining a bro~d a83ay r~nga.
Using the calibration cur~e of Fig 6, a detection ~en~itivity of l~g/ml C reactiYe protein can b~
achieved. The C-reactive protein concentrations in 11 human 3era have been correctly mea~ured by the assay of the invention as indicated by comparison to a FDA-approved reference procedure. The correlation curve i~ shown in FigNre 7.
It i~ evid~nt fro~ the above reault~ tha~ the sub~ect ~e~hod pro~ides for an accurat~, sen~itive, and rapid technique for detecting levels of high-molecular-weight analytes, particularly in a complex 2 ~ 7 ~i ~ixt~re. The ob~erved en~yme actlvity i~ inv~r~ely proportioll~l to the amount of analyte ln the 8alllple.
All publication~ and p~tent applicstionE cited in thi~ speci~icatlon are herein ~ncorporated ~y 5 reference a~ if each individual public~t:Lon or patent application were ~pecifically and individually indicated to be incorporated by refer0nce.
Although tlle foregoinq invention ha~ b~en de~crib~3d in ~ome detAil by way of illustration ~nd 10 exainple for purpo~e~ of clarity of urlder~tanding, i~
will be readily appar~nt to tho~e of ordillary ~k~ 11 in the art in light of the teaching~ of this inven'cion that certain change~ and modific~tions can be made ther2to without departing from the ~pirit or ~cope of 15 the appended claim3.
~1
Claims (23)
1. A method for detecting the presence of a high-molecular-weight analyte using a .beta.-galactosidase complementation assay, comprising:
(a) providing an enzyme donor (ED) complex comprising ED coupled to an analyte-specific binding moiety, wherein said ED complex retains measurable complementation activity such that active .beta.-galacto-sidase is formed in the presence of enzyme acceptor (EA);
b) contacting the ED complex and EA in the presence of a sample suspected of containing a high-molecular-weight analyte that reacts specifically with said analyte-specific binding moiety, and (c) relating the presence of the analyte in the sample to the formation of active .beta.-galactosidase enzyme.
(a) providing an enzyme donor (ED) complex comprising ED coupled to an analyte-specific binding moiety, wherein said ED complex retains measurable complementation activity such that active .beta.-galacto-sidase is formed in the presence of enzyme acceptor (EA);
b) contacting the ED complex and EA in the presence of a sample suspected of containing a high-molecular-weight analyte that reacts specifically with said analyte-specific binding moiety, and (c) relating the presence of the analyte in the sample to the formation of active .beta.-galactosidase enzyme.
2. A method according to Claim 1, wherein said ED complex, EA, and sample are contacted with an enzyme substrate which provides a measurable product upon reaction with .beta.-galactosidase, the measurable product provided by the reaction of the enzyme substrate and active enzyme is measured, and the amount of said analyte in the sample is determined by comparing the amount of measurable product to that formed from a known amount of analyte.
3. A method according to Claim 2, wherein excess unlabelled polyclonal or monoclonal antibody specific for said analyte is also present when said ED complex is contacted with EA in the presence of said analyte.
4. A method according to Claim 1, wherein said analyte is a polypeptide.
5. A method according to Claim 1, wherein said analyte ha a molecular weight of at least 10,000.
6. A method according to Claim 1, wherein said analyte has a molecular weight of at least 20,000.
7. A method according to Claim 1, wherein said analyte is a nucleic acid.
8. A method according to Claim 6, wherein said nucleic acid is DNA.
9. A method according to Claim 6, wherein said nucleic acid is RNA.
10. A method according to Claim 1, wherein said analyte is a virus particle.
11. A method according to Claim 1, wherein said binding moiety is an antibody or a natural receptor
12. A method according to Claim 9, wherein said antibody is a monoclonal antibody or recombinant antibody or recombinant peptide fragment equivalent of an antibody.
13. A method according to Claim 6, wherein said binding moiety is a nucleic acid DNA or RNA.
14. A method according to Claim 2, wherein said substrate is CPRG.
15. A method according to Claim 1, wherein said ED is linked to an antibody specific for said analyte by a bifunctional organic linking group.
16. A method according to Claim 1, wherein ED-binding moiety is a recombinant fusion protein.
17. A kit for use in detecting the presence of a high-molecular-weight analyte in a sample employing complementary fragments of .beta.-galactosidase, which fragments are defined as enzyme donor (AD) and enzyme acceptor (EA) and when bound form active .beta.-galactosidase, which kit comprises:
an ED complex comprising ED covalently attached to a binding moiety, and EA, wherein said EA and said ED complex are present in one or more containers.
an ED complex comprising ED covalently attached to a binding moiety, and EA, wherein said EA and said ED complex are present in one or more containers.
18. The kit according to Claim 14, which kit further comprises an enzyme substrate which provides a measurable product upon reaction with .beta.-galactosidase and allows for the determination of the amount of said analyte in the sample by comparison with the amount of measurable product formed from a known amount of analyte.
19. A kit according to Claim 15, wherein Raid binding moiety is an antibody.
20. A kit according to Claim 15, wherein said ED is linked to said antibody by a divalent organic linking group.
21. A reagent complex, comprising an enzyme donor (ED) fragment covalently attached to a binding moiety specific for an analyte and having a molecular weight of at least 10,000 but less than 2,000,000, wherein said ED forms active .beta.-galactosidase upon being brought into contact with its complementary enzyme acceptor fragment in a complementation assay.
22. The reagent complex of Claim 18, wherein said binding moiety is an antibody or nucleic acid.
23. The reagent complex of Claim 18, wherein said binding moiety is a monoclonal antibody covalently linked to said ED by a bifunctional organic organic linking compound.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/649,483 | 1991-02-01 | ||
| US07/649,483 US5244785A (en) | 1991-02-01 | 1991-02-01 | Determination of high molecular weight analytes using a β-galactosidase complementation assay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2060377A1 true CA2060377A1 (en) | 1992-08-02 |
Family
ID=24604988
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002060377A Abandoned CA2060377A1 (en) | 1991-02-01 | 1992-01-30 | Determination of high molecular weight analytes |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US5244785A (en) |
| EP (1) | EP0497613B1 (en) |
| JP (1) | JP2892879B2 (en) |
| AU (1) | AU653196B2 (en) |
| CA (1) | CA2060377A1 (en) |
| DE (1) | DE69211371T2 (en) |
| ES (1) | ES2090492T3 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5612019A (en) * | 1988-12-19 | 1997-03-18 | Gordon, Deceased; David | Diagnosis and treatment of HIV viral infection using magnetic metal transferrin particles |
| CA2068190C (en) * | 1991-05-15 | 1996-12-17 | Microgenics Corporation | Methods and compositions for enzyme complementation assays using the omega region of beta-galactosidase |
| DE4310141A1 (en) * | 1993-03-29 | 1994-10-06 | Boehringer Mannheim Gmbh | Homobidental trifunctional linkers |
| US5427912A (en) * | 1993-08-27 | 1995-06-27 | Boehringer Mannheim Corporation | Electrochemical enzymatic complementation immunoassay |
| US5792617A (en) * | 1994-08-18 | 1998-08-11 | Rotman; M. Boris | Cell proliferation-based amplified detection of analytes |
| JP2014505059A (en) | 2011-01-18 | 2014-02-27 | ジェネラル アトミクス | Hydrolase enzyme substrate and use thereof |
| WO2019104529A1 (en) * | 2017-11-29 | 2019-06-06 | 深圳琅技生命科技有限公司 | Homogeneous phase detection method |
| WO2019171967A1 (en) * | 2018-03-07 | 2019-09-12 | コニカミノルタ株式会社 | Specimen diluent, method for preparing sample, sample, and sandwich method |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2334107A1 (en) * | 1975-12-05 | 1977-07-01 | Pasteur Institut | METHOD OF COUPLING BIOLOGICAL SUBSTANCES BY COVALENT BONDS |
| US4708929A (en) * | 1984-10-29 | 1987-11-24 | Microgenics Corporation | Methods for protein binding enzyme complementation assays |
| WO1986002666A1 (en) * | 1984-10-29 | 1986-05-09 | Microgenics Corporation | Methods for protein binding enzyme complementation assays |
| AU610281B2 (en) * | 1987-09-21 | 1991-05-16 | Roche Diagnostics Corporation | Solid-phase non-separation enzyme assay |
| US4994385A (en) * | 1987-10-30 | 1991-02-19 | Abbott Laboratories | Heterobifunctional coupling agents |
| US5037735A (en) * | 1988-06-24 | 1991-08-06 | Microgenics Corporation | Visual discrimination qualitative enzyme complementation assay |
| SE8901566D0 (en) * | 1989-04-28 | 1989-04-28 | Ferring Diagnostica Ab | NEW PEPTIDES INCLUDING A DISULFID BOND |
| WO1990013569A1 (en) * | 1989-05-05 | 1990-11-15 | Microgenics Corporation | Method for protein binding enzyme complementation assays |
-
1991
- 1991-02-01 US US07/649,483 patent/US5244785A/en not_active Expired - Fee Related
-
1992
- 1992-01-30 CA CA002060377A patent/CA2060377A1/en not_active Abandoned
- 1992-01-31 JP JP4016386A patent/JP2892879B2/en not_active Expired - Fee Related
- 1992-01-31 ES ES92300829T patent/ES2090492T3/en not_active Expired - Lifetime
- 1992-01-31 EP EP92300829A patent/EP0497613B1/en not_active Expired - Lifetime
- 1992-01-31 DE DE69211371T patent/DE69211371T2/en not_active Expired - Fee Related
- 1992-01-31 AU AU10687/92A patent/AU653196B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| DE69211371D1 (en) | 1996-07-18 |
| AU653196B2 (en) | 1994-09-22 |
| JP2892879B2 (en) | 1999-05-17 |
| AU1068792A (en) | 1992-08-06 |
| ES2090492T3 (en) | 1996-10-16 |
| US5244785A (en) | 1993-09-14 |
| EP0497613A1 (en) | 1992-08-05 |
| DE69211371T2 (en) | 1996-11-28 |
| EP0497613B1 (en) | 1996-06-12 |
| JPH0566223A (en) | 1993-03-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0782710B1 (en) | Method for detecting antibodies | |
| EP0347138B1 (en) | Specific binding composition comprising a low pI protein or carbohydrate and a diagnostic test kit and method of use | |
| EP0992794B1 (en) | Enzyme-antibody complex and a method for manufacturing the same | |
| FI73322B (en) | FOERFARANDE FOER BESTAEMNING AV KARCINOEMBRYONAL ANTIGEN OCH DETTA FOERFARANDE ANVAENDBAR LOESNING. | |
| EP0315364A2 (en) | Simultaneous immunoassay for the determination of antigens and antibodies | |
| KR101158271B1 (en) | Probe complex | |
| CA2060377A1 (en) | Determination of high molecular weight analytes | |
| CN113866406B (en) | Kit for specifically detecting sugar-deficient transferrin | |
| Burgett et al. | A solid phase fluorescent immunoassay for the quantitation of the C3 component of human complement | |
| EP3832306A1 (en) | METHOD FOR IMMUNOLOGICAL ANALYSIS OF (1->3)-ß-D-GLUCAN IN BIOLOGICAL SAMPLE, KIT FOR ANALYSIS OF (1->3)-ß-D-GLUCAN, AND ALKALI PRETREATMENT SOLUTION FOR BIOLOGICAL SAMPLE FOR USE IN METHOD FOR IMMUNOLOGICAL ANALYSIS OF (1->3)-ß-D-GLUCAN | |
| CA2073452A1 (en) | Combination assay for antibody or antigen | |
| EP0338045B1 (en) | Solid-phase non-separation enzyme assay | |
| JP3667434B2 (en) | Nonspecific reaction inhibitor used for immunoassay, nonspecific reaction suppression method and measurement kit | |
| JPS583671B2 (en) | Methods of increasing the ability of apoglucose oxidase to bind flavin adenine dinucleotide and its derivatives, homogeneous specific binding assays, homogeneous immunoassays for measuring complexes and ligands in liquid media, Reagents and | |
| CA2028146A1 (en) | Composition containing labeled streptococcal antibody, test kit and assay using same | |
| CN110234995A (en) | Use the immunoassays of the analyte specificity combinating reagent of at least two Pegylations | |
| US5212064A (en) | Solid phase non-separation enzyme complementation assay | |
| US5998157A (en) | Acylated protein aggregates and their use as signal enhancers in an immunoassay for the detection of antibodies | |
| JP3667435B2 (en) | Non-specific reaction inhibitor, suppression method and measurement kit | |
| CA1335174C (en) | Solid-phase non-separation enzyme assay | |
| WO1993022325A1 (en) | Immunoassay for 3', 5' cyclic adenosine monophosphate | |
| EP0184701A2 (en) | A method for determining a ligand | |
| Note | Contaminant human transferrin assay | |
| Note | Contaminant bovine transferrin assay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |