CA1308652C - Agglutination assay - Google Patents
Agglutination assayInfo
- Publication number
- CA1308652C CA1308652C CA000577502A CA577502A CA1308652C CA 1308652 C CA1308652 C CA 1308652C CA 000577502 A CA000577502 A CA 000577502A CA 577502 A CA577502 A CA 577502A CA 1308652 C CA1308652 C CA 1308652C
- Authority
- CA
- Canada
- Prior art keywords
- analyte
- erythrocyte
- binding molecule
- antibody
- reagent
- 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.)
- Expired - Lifetime
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
- G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
-
- 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
- G01N33/531—Production of immunochemical test materials
-
- 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
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56988—HIV or HTLV
Abstract
Abstract In a novel, erythrocyte agglutination assay, the agglutination reagent comprises an erythrocyte binding portion attached to a specific analyte binding portion or to an analyte analogue wherein the reagent does not cause agglutination when incubated with endogenous erythrocytes in the absence of analyte or analyte binding reagent. Mixtures of reagents may also be used as agglutination reagents. The reagents and their use in direct or indirect assays is disclosed.
T?/148?
T?/148?
Description
-````` 1 308652 ERYTHROCYTE AGGLUTINATION ASSAY
TECHNICAL FIELD
.
The present invention relates to a reagent and a method for detecting an antigen, antibody or other analyte in human or animal blood by erythrocyte agglutination. The invention also concerr,s a kit containing the reagent and processes of preparation of the reagents.
BACKGROUND ART
Assaying blood samples for a particular antigen or antibody has traditionally involved the step of separating the cellular components from the serum components of the blood by centrifugation and/or clotting, prior to assay.
This presents several potentlal problems. Firstly, such an assay is not suited to testing being conducted under field conditions. In many veterinary situations a quick test in the field i5 more desirable than the alternative of transporting samples to laboratories for separation and assay. Also, veterinary surgeons who do not have access to a centrifuge frequently need to assay blood samples for the presence of infectious agents such as heartworm. Further, assays being used for the detection of diseases in Third World countries present a siiuation where simplicity and low cost are of the essence.
Secondly, in certain pathologic conditions, separation of the blood samples becomes difficult. Blood taken from patients suffering conditions such as Waldenstrom's macroglobulinemia is difficult to separate into serum ancl cell fractions making an assay which can be conducted on whole blood highly desirable.
Thirdly, blood samples are often used for testing tor the presence of highly contagious and potentially dangerous disease states. In these cases it is preferable that as little handling and processing of the samples as ~k TLH/148c - 1 -possible is undertaken in order to minimize the risk to personnel conducting the assay. Further, certain conditions make the provision of over-the-counter finger-prick assays highly desirable. Such assays must necessarily be suited to performance on whole blood.
Immunoassays have revolutionized human diagnostic and veterinary medicine since the introduction of techniques such as the radioimmunoassay, first reported by Yalow and Berson (1959) Nature 18~, 1648, and the enzyme immunoassay or EIA which was first reported by Engvall and Perlman (1971 Immunochem 8, 871 and Van ~eeman and Schuurs (1971) FEBS Letters 15, 232.
Whilst such assays are based on antibody-antigen interactions the detection systems utilized are usually complex. The reagents used are generally enzyme or radiolabelled antigens, antibodies or complexes thereof which require either incubation with specific substrates and measurement of a color end-point either visually or by means of a colorimeter or measurement of radioactive decay with radiation counters to detect the presence of the analyte being tested. These assays also in~olve several washing steps. Most immunoassays for the detection of analytes in blood are currently of this nature. Thus, whilst these assays are sensitive, they are lengthy and involve procedures which may require expensive instrumentation, for detection of the analyte under test.
An alternative to these assays is provided by immunoassays of the A type described by Gupta, et al., (1985) Journal of Immunological Methods 177-187. These are immunoassays in which erythrocytes and anti-erythrocyte antibodies are used in the indicator system. In these assays exogenous erythrocytes such as sheep erythrocytes are used.
In recent years it has been possible to attach antibodies tO latex beads, thus providing a rapid agglutination assay. This, however, still entails the separation of the serum/plasma phase from the cellular phase TLH/148c - ~ -and consequently requires the use of a centrifuge or filtration system.
Latex agglutination assays are described in Castelan, et al., J. Clin.
Pathol. (1968), 21, 638; and Singer & Plotz AM. J. Med.
~1956 (Dec)~, 888.
Both direct and indirect agglutination immunoassays are well known in the art. In these assays, the agglutination of particles to which antigen or antibody is bound is used to indicate the presence or absence of the corresponding antibody or antigen. A variety of particles, including particles of latex, charcoal, kaolinite, or bentonite, as well as both microbial and red blood cells, have been used as agglutinatable carriers.
See Mochida, US 4,308,026. The use of erythrocytes as indicator particles is strongly criticised by Patel, U5 3,882,225, who says that it is difficult to standardize indlcator erythrocyte 5.
Molinaro, US 4,130,634 describes an assay for an antigen which employs antibody-cc~ated red blood cells. Molinaro emphasizes that the method used to couple the antibody to the erythrocyte must not destroy the reactivity of the antibody. He makes it clear that antibodies which are specific for the erythrocyte are not useful for his assay. He does mention, however, the posslbility of using a hybrid antibody with one binding site specific for the antigen and the other specific for the red blood cell.
Chang, US 4,4~3,05~ discloses an agglutination immunoassay reagent in which two antibodies are covalently linked "ta~l-to-tail", i.e., so as not to alter their specificity. One antibody is specific for an antigen borne by an indicator substance, such as an erythrocyte. This antibody i5 preferably un~valent to avoid nonspecific agylutination. The other antibody is divalent and is speclflc for the analyte. In preparation for the assay, fresh erythrocytes are coated with the conjugate. The double TLh/148c - 3 -1 30~652 antibody conjugate-coated RBCs are then incubated ~Jith the test serum.
Chang does not contemplate the assaying of whole blood samples using a non-autoagglutinating anti-RBC antibody and endogenous erythrocytes.
Chu, US 4,493,793 discloses the construction of a lectin-antibody or lectin-antigen covalently coupled conjugate. His Table I (incorporated by reference) sets forth the carbohydrate specificities of several lectins.
He does not teach coupling such a conjugate to an erythrocyte through either the lectin or the antibody receptor.
Other "tail-to-tail" immunological conjugates are known. Segal, US
4,676,980 sets forth the construction of a "tail-to-tail" conjugate of a target cell surface antigen-specific antibody and of a cytotox~c effector cell receptor-specific antibody. Several cross-linking methods, incorporated by reference, are described. This conjugate is intended for use in immunotherapy, in that it will cause the cellular immune system of the patient to lyse the target cell. The target cell would not, of course, be an erythrocyte endogenous to the host.
Li, US 4,661,444 suggests the production of a tail-to-tail conjugate of an analyte-binding antibody and of an antibody specific for the idiotype of the first antibody. This conjugate was to be used in conjunction with an insolubilized analyte-binding antibody in an immunoassay.
~ ardlaw, US 4,695,553 teaches use of a monoclonal antibody against a universal erythrocyte antigen as a RBC ag~lutinating agent to clarify the interface between red blood cells and white blood cells in centrifuged whole blood. ~le prefers use of antibodies against glycophorin or against antigen, but also mentions the posslbility of using a mixture of lectins.
Guesdon, US 4,668,637 discusses the use of anti-red blood cell antibodies or of lectins for the purpose of erythroadsorption. Bigbee, [Molecular Immunology, ~0: 1353-1362 (1983)] describes the production and testing of TLH/148c - 4 -four monoclonal antibodies against glycophorin A. The general concept of using in an immunoassay an antibody which reacts with an antigenic determinant shared among all members of a class of analytes of interest (microorganisms) is set forth in McLaughlin, US 4,683,196.
A number of patents deal with antibodies useful in blood typing.
See, e.g., Lloyd, US 4,678,747; Graham, Jr., US 4,358,436; Liu, US
4,550,017; Steplewski, US 4,607,009; Lennox, W083103477. Tnese antibodies are useful for blood typing because they bind to antigens found only in certain blood cell populations, while for the purpose of this invention, it is desirable to use antibodies (or mixtures thereof) which bind to essentially all erythrocytes.
Zuk, US 4,594,327 recognizes the desirability of performing an immunoassay directly on whole blood samples. In his method, the sample is contacted with both an insolubilized, analyte-specific immunoreagent and with a red blood cell binding agent such as a RBC-specific antibody or a lectin. The analyte-specific immunoreagent and the ReC binding agent are not coupled together, and the assay disclosed is not an agglutination assay.
The problem, in an agglutination immunoassay, of nonspecific agglutination of erythrocytes by anti-erythrocyte antibodies endogenous to the blood sample, was noted by Czismas, US 3,639,558. He proposed eliminating all naturally occurring antigenic sites on the particle by coating the particle with protein.
Theofilopoulos, US 4,342,566; Duermeyer, US 4,292,403 and Goldenberg, US 4,331,6q7 are of interest as demonstrating the use of specific binding fragments of antibodies as substitutes for intact antibodies in assays.
The construction of heterobi~unctional antibodies is taught by Auditore-Hargreaves, US 4,446,233; Paulus, US 4,444,878; and Reading, US
4,474,893. Mochida, US 4,200,436 discloses the use of monovalent TLH/148c ~ 5 ~
antibodies or binding fragments thereof in certaln lmmunoa~says Forrest, US 4,65~B78 mentions that monovalent antlbodies cannot form dlmers or more extens1ve complexes with thq ant~en; such aggreg~tes w~re said to be c~pable of interfer1ng with ~he bind~ng of the antlgen-antibody complex to a solid phase support.
According to a flrst embodiment of thls lnvention there is prov~ded an agglutinatlon reagent for dlrect detcction of an analyte ln a blood sample, compris~ng an erythrocyte bindln~ molQcule attach2d to an analyte bindin~ molecule, whereln the erythrocyte bindlng molecule is attached to the analyte b~nd1ng molecule in such a manner that the blndlng ch~ractertstics of the ~tnding molecules are not altered by the attachment, and the erythrocyte blnding molecule 1s capable of bind~ng erythrocytes ~ndogenous to the blood sample but does not agglutina~e endogenous erythrocytes in the absence of analyte, wtth the proviso that when sald ery~hrocyte blndlng m~lecule ls an anti-erythrocyte-ant~body or part thereof, s~d analyte blnding mvlecule ls no~ an ant~body attached thereto by means of a he~srobl~u~ctlon~l couplln~ r~agent.
Acco~d~ng to a s~cond embodiment of this lnvention ther~ ls pr~vlded an agglutlnatlon reagent whlch co~pris~s a conJuga~e comprislng at least one erythrocyte bindtng molecule con~ugatcd with at least on~
~nal.yte b~nding molecule, sa~d cc,n~ugate agglutinatlng erythrocy~s essentlally only ln the presence o~ the analyte~ whereln sald con~ugate does not substantially alter the bindtng characteristlcs of satd erythrocyte bindlng molecule and sald analyte binding molecule or lyse said erythrocytes; and wherein satd erythrocyte ~nd~ng mol0cule ~s a i~tl-ulllvdlulll duLl-~lythl~cyto ~ntlbo~y, ~hereln ~Ql~ ~nttbody or fragment ~ss~nt~ally does no~ aut~-a~lut1n~te erythrocytes, or a ."~ .
fragment of such ~n ~ntibody .
According to a third embodlment of th~s In~ent10n there ls prov~ded an agglutlnatlon reagent for assaying analyte whlch comprlses a heterobifunctional antibody or a heterobl~unct~onal b~nd~n~ fra~ment of an ant~bo~y, said antibody consistl ng of an erythrocyt~ ~lnd~ng molecule which ~lnds erythrocytes but not the analyte, and an analyte blndln~
molecule b1nd~ng the analyte but not erythrocytes, the erythrocyte blndlng molecule belng con~ugatcd to the analyte blnding molecule by one or more dlsulphldQ bonds and not by a heteroblfunctlonal eoupllng agent.
Accordlng to a fourth e~bodiment of this invent10n there 1s provided an agglutlnation reagent whlCh comprlses a coniugate comprls~ng an erythrocyte b~ndlng molecule con~ugat~d w1th an analyte blndlng m~lecule whereln the erythrocyte blndlng molecule is a peptide hav~ng an a~finity for the erythrocyte membrane but lncapable Gf lyslng erythrocytes, and ls not derived from an antlbody or leetln~
Accordlng to a ~l~th embodlment of this in~ention there lS proY1ded a d1rect agglut~nation assay for the presenee of an analyte ln a blood ~ampl~ contain~ng erythrocytes whgch assay comprlses mlxlng the sample wl~h an agglutlnation reag~nt accordlng to any of the ~lrst to fourth embod~ents and obsQrvlng whether the erythr~cytes are a~glutlnated and correlatln~ the ag~tutinat~on w7th tne amount of analyte present 1n ~he sampl e .
Accordlng to a slxth embodiment of thls lnventlon there is provlded a dlrec~ agglutlna~lon assay ~or the presence or amoun~ of an analyte ln a sample whlch comprlses forming a mlxturQ of a sample, erythrocytQs~ and an agglutlnatlon reagent whlch reagent comprises a con~ugate ~ompris~ng an erythrocyt~ bindlng molecule con~ug2ted wlth an analyte b~ndlng molecule, wh~re1n th~ erythrocyte b~ndln~ mo~ecule is a p~ptlde h~ving an aff~n~ty for ~he erythrocyte membrane but incapable of lysinig erythrocyt~s, and ls not d~rlved from an ant~body or l~ctln; ~bservln~
wheth~r the erythrocytes are agglutlnatQd and dlrectly correlat~ng the presence or amount oP agglutlnatlon wlth the presence or amount o~
analyte present.
Accordlng to a seventh ~mbodlment o~ thls lnventlon there ls provided a d~rect agglutlnation assay for the presence of an analyte ln a whole blood çample from a sub~ec~ which comprises contRctlng a blood samp7e containtng erythrocytes endogenous to the sub~Qct w1th an agglutination reagent whlch compris~s a conjugate of (a) a multivalent monoclonal antlbody which blnds to ~rythrocyte membranes or a b~nd1n~
~ragment thereof, and (b) an analyte blndlng molecule, sald con~ugate belng essentl~lly lncapable of agglutinatlng sald erythrocytes ln the abs~nce of analyte, observing wheth~r th~ ery~hrocytes are ag~lutlnated and dlrectly correlat1n~ the ~gglutlnatlon w~th the presence or amount o~
analyte present.
Accordlng to an e~ghth e~o~lment of th1s lnventlon thQre ls prov~d~d an agglutlnatlon ass~y for an analyte laeklng repeat~ng epitope~
wh~ch compr1s~s ~ncubattng a sample whlch i~ay contaln such analyte, erythrocytes, a con~ugate o~ an erythrocyte blnding molecule and an analyte b1ndlng molecule, and a non-unlva~ent secondary b~ndlng molecul~
whlch binds to a new epltope formed by the blnding of the analyte blndlng molecule to the analyte, and correlat1ng the presence or d~gree of agglutlnation wlth the presence or quantlty o~ the analyte ln the sample.
Accordlng to a nlnth embodiment o~ this inventlon there ls provl~ed an agglutlnatlon assay ~or an analyte lackln~ repeat1ng epl~opes whlch comprises lncubatlng a sample which may contain such analyte, erythrocytes~ a con~ugate of an erythrocyte bindlng molecule and an ,, ~ .
,r .
analyte blnding molecule, where sa~d con~ugate essentlally does not auto-agglutinate-erythrocytes, and a secondary btndlng molecule which binds to a new epitope formed by thQ blnding o-f the analyte btndlng mo~ecule to tne analyte, and correlatlng the presence or d~gree of agglutinatlon wlth the presence or quantity of the analyte tn the sample, wheretn the secondary bindlng molecule ls also conju~ated to an erythrocyte blndSng molecule.
Accordlng to a tenth embodimen~ of thls tnv~ntlon there ls provlded a dlrect ag~lutlnation assay for the presence or amount of an analyte ~n a sample whlch comprtses forming a m~xture of a sample, erythrocytes7 and an agglutlnatlon reagent whlch comprlses a conju~ate comprls7ng an erythrocyte bind~ng molecule con~ugated wlth an analyt~ btndln~ molecule whereln the con~u~ate ls a heterobtfunc~10nal antlbody or a he~erobifuncttonal blnd1ng fra~ment o~ an antibody, said antibody conslsttn~ of an erythrocyte bindtn~ ant1body fragmQnt whlch blnds erythrocytes but not the an~lyte, and an analyte binding anttbvdy fra~ment blnding th~ analyte but not erythrocytes, the ~rythro~yte binding frag~ent b~ing conjugated to the analyt~ b1ndlng fl~g~Qnt by one or mDre dtsulphlde bonds and not hy a heterobtfunct~onal coupling agent, and wh~reln the reagen~ co~prlses a detectabl~ amount of homo~lfuncttonal erythrocyte-blndlng ant{body, but sald ho~oblfunctlonal erythrocyte-blndlng ant~body essentially does not auto-agglu~lnat~
erythrocytes.
Accordlng to an eleventh Qmbod~ment of thls invention there ~s prov1ded an agglutinat~on reagent for ~ndtrect de~ectton of an analyte ln a blood sample, c~mprlslng an erythrocyte blndlng molecule attached to an analyte analo~ue wherein satd erythroeyte binding molecule ts a~tached to salct analyte analogue in such a mannQr that blnding characterlsttcs o~
. .
the erythrocyte blnding molecule are not altered by the attachment, and said erythro~yt~ blndlng molecule is capable of binding erythrocytss endogenous to the sample but does not agglutlnate endogenous erythrocytes 1n the absence of an analyte blndlng reagent.
According to a twel~th embodlment of this invention ther@ 15 provided an agglutlnatlon reagent which comprlses a con~ugate c~mprls~ng an erythrocyte blndlng molecule conjugated wlth an analyte analogue where1n sa1d con~ugate does not substantlally alter the bindlng characteristlcs of the erythrocyte b~nding molecule (EBM), or the analyte analogue and does not lyse erythrocytes, wherein sald conjugate essent~ally does not agglutlnate erythro~ytes ln the absenc~ of an analyte binding rQagent~ and wherein sald EBM ls a non-unlYalent anti-ery~hrocyte ant1body or a ~ra~ment of such an antlbody whereln sald ant1~0~y or fragment essentlally do not auto-a~glutinate erythrocytes.
Accord~ng to a twelfth embodim~nt o~ th~s lnventlon there 1s provlded an agglutlnatlon reagent which comprlses a son~ugate comprising an erythrocyt~ bind1ng molecule conjug~d w1th an analyte analogu~, whQreln the erythrocyte b1nding ~olecule ls a p~ptlde havlng an aff1nlty for the erythrocyte membrane ~ut ~ncap~bte o~ lys1ng erythrocytes, and ts not ~er1vQd from an ant~body or lectin.
Accor~lng to a thirteenth embodiment of th~s ~nvont10n there ts provided an 1nd1rec~ agglut1natlon assay for the presence or amount of an analyte in a blood sample whlch comprises ~ncubatlng the sample w~th an agglut~na~lon reagent accordlng to the eteventh or twelfth embod~ment and a soluble ana1yte blnding reagent, whereby said agglutinatlon reag~nt compQte~ wlth sample analyt~ for the analyte blnding sltes of sa1d analyte blndlng reagent observing whether a~glutinat~on occurs, and determinlng the presence or am~unt of sa1d analyte from the lnverse o~
,~ ' the degree of ag~lut~nation.
Accordlng to~ fourteenth embodlmen~ of th~s invent~on there ls prov1ded an ind1rect agglutlnation assay for the presence or amount of the analyte in a sample which comprlses ~a) formln~ a ~xture of sample, erythrocytes, an agglutlnatlon reagent whSch comprises a con~u~ate compristng an erythrocyte binding molecule conjugated w~th an analyte binding molecule, wherein the erythrocyte b1ndln~ molecule is a peptlde having an a~finity for the erythrocyte membrane but lncapable of lysing ery~hrocytes, and 1s not derived from an antlbody or lect1n, and a soluble non-unival~nt analyte b~nding rQagent which is essentially ~ncapable on ~ts own of agglutlnating erythrocytes, (b) permttt~ng sa~d conjugate to compete with sampl~ analyte for the analyt~ ~lnd~ng sites o~
the analyte b1nd~ng reagen~, ~c) observing whether agglut1nation o~curs, and ~d) inversely correlating the degree of agglutinatlon w~th the amount of analyte pres~nt.
Accord~ng to a fl~tQenth embodlment of this invent~on there ls provided an ln~lrect ag~lutlnation assay for the presence o~ an analyte ~n a whole bl~od sample from a s~b~ect which comprises (a) contactln~ a blood sample containing erythrocytes endogenous to the sub~ect w1th Sl) an agglutlnation rea~ont wh~ch com~r~s~s a conjugate of an ~ntact multlvalent monoclonal ant1~ody wh~ch binds to erythrocyte me~ranes or a ~inding fragment thereo~, and an analyte analogue, satd con~ugate belng eapable of agglutinat1ng erythrocytes but only ln the presence of a mul~ivalent analyte blnding agent, and (iiS a soluble analyte blndln~
reagent wh~eh ~s essen~ially 1ncapable of agglu~inating erythrocytes, (b) permitting sa1d conjugat~ to compete with sample analyte for the analyte b~nding sites o~ ~he analytq b1nding rea~ent, ~c) observlng whether aggl~tlnatlon occurs, and ~dS inversely correlat~ng the degree o~
1 30~652 agglutlnat10n w1th the presence or amount of analyte present.
According to a sixteenth embodiment of thls inventlon there 1s p~ovlded a test kit for use 1n dlrect ag~lutlnatlon assays which compr~ses (a~ a con~u~ate of an ery~hrocyte b1nd~ng ~olecule and an analyte blnd1ng molecule ~al d con~ ugate be 1 ng capable of agglutlnating erythrocyte~ only ln the presence of the analyte and (b) a seeondary btndlng mole~ule whlch b~nds to a new epitope ~ormed by the blnd~ng of the analyte bindlng molecul~ to the analyte.
According to a seventeenth e~bod1ment of this 1nvention there ls provlded a dlrect agglu~lnation assay test k1t comprts~ng an agglutlnation reagent according to any one of the first to fourth embodiments and a reference solut~on contalning a known quantity of analyte.
Accord~ng to an eighteenth embodiment of ~hls inven~on there ~s prsvided a t~st kit for use ~n 1ndlrect ~gglut~nat10n assays which comprlses an aggluttn~tlon reagent according to the elevQnth or ~wel~h embod1men~ and a soluble analyte blndlng reagent.
The present tnventors reco~n~zed that there was a nced ~or a me~hod whtch can be us~d in the laboratory and 1n th~ f1eld part1cularly ln Thlrd World Countr~es where there is l~ck of medical testin~ ~ciltties for analysls of d1~ferent typ~s of ana1ytes in whole blood. As ~ndlca~2d above earli~r meth~ds requlre scpar~tlon of the blood cells from serum or plasma and are ther2fore d1fflcult and in many cases ~mposslble to ~mplement ~n the fleld.
If erythrocyte-bindlng molecules are coupled to sp2clflc analyte~
b1ndin~ mol~cules then the r~sulting con~ugate could be used to blnd both Ln53Qçn9u5 erythrocytes and analytes present 1n a blood sample.
The present tnventlon resul~s from the flndlng that when such a complex ... - 6F -, ~ ' 1 30~652 ls exposed to a blood sample, agglutlnatl~n of the erythrocytes endogenous to tha~.sample will serve as an Indicator of the pres~nce of the retevant analyte (usually, an antigen or antibody) due to cross-l~nklng of erythr~cytes wlth the analyte.
Adv~ntages of endogenous RBCs O ~
- no need to centrlfuge sample whole blo~d, collected in the presence o~ a suitab1e anticoagulant, is used instead of seru~
or pla~a.
- for samples ~rom patients with in~ectious diseases, such as AIDS or hepatit~s, there ~s mlnimal sample handling.
-/.
/
-, . ..
1 30~652 appropriate for mass screening programs as conducted by the ~orld Health Organization in third world countries, whose facilities are limited.
the assay is very robust; there is only a single reagent, which is stable in the presence of a bacteriostatic agent such as 0.01% (w/v) sodium azide.
can be used as a field test by veterinary practitioners, when the appropriate animal red cel1s are used for immunization to produce species specific MAb.
the test is very fast - agglutination occurs in less than three minutes.
the method can be used to monitor therapeutic drugs and patient compliance.
it also has possible use as an "over-the-counter" self testing assay.
the only equipment needed is a mixin~ stirk, glass or plastic slide, lancet and possibly a microcapillary.
Advantaqes over exoqenous ervthrocytes include:
no pretreatment of erythrocytes. Patent 4,433,059 issued February 21, 1984 to Chi-Deu Chang and ~enry A. Graham uses blood group O
negative cells, which have been spun down, reacted with antibody conjugate for 15-30 minutes and washed 3x in PBS. Patent 4,668,637 issued May 26, 1987 to Jean-Lue Guesdon and Stratis Avrameas uses sheep red blood cells, which have been washed and resuspended in PBS. After the reaction, which takes place on a solid support, the cells are then fixed.
no pretreatment of samples. Patent 4,433,059 (identified a~ove) notes that samples have to be heat inactivated to avoid interference due to complement. Rabbit serum and bovine albumin must also be added to minimize other non-specific reactions. None of this is 148c , .
.
necessary with the present systern, where undiluted whole blood from patients may be reacted directly with reagent. This reagent contains unrelated monoclonal antibody to prevent any anti-mouse reactions, which may occur.
Thus, it is possible to dispense with the cumbersome separation of cells from serum and with the sensitization and fixing of exogenous erythrocytes intended for use as indicator particles in agglutination assays. The endogenous erythrocytes are sensitized by the reagent.
Another novel aspect of applicants' agglutination reagents and assays is the selection of an erythrocyte binding molecule such that incubation of conjugate with endogenous erythrocytes will not cause agglutination of the erythrocytes unless analyte is also present -- such erythrocyte binding molecules are termed herein "non-auto-agglutinating~.
The erythrocyte binding molecule is preferably a monoclonal antibody and especially, in the human system, an anti-glycophorin antibody. It is believed that this antibody is non-autoagglutinating for steric reasons;
either the binding sites of the intact antibody are able only to bind adjacent epitopes on the same erythrocyte or only one of the two binding sites can bind to glycophorin at one time.
Applicant's assay can detect small antigens without repeatlng determinants, using two conjugates, one bearing an analyte-specific binding molecule and the other, a binding molecule specific for a new epitope formed by the binding of the first conjugate to the analyte. This allows cross-linking in the presence of the antigen to be measured.
Brief Description of the Drawinqs Fig. 1 is a schematic representation of erythrocytes showing positive and negative agglutination results with antibody complexes in the presence and absence of antigen respectively.
TLH/148c - 8 -``` 1 308652 Fig 2. is a schematic representation of erythrocytes showing positive and negative agglutination results with a complex of antibody and an antigen in the presence and absence, respectively of anti-antigen antibodies.
Fig 3. is a schematic representation depicting (a) erythrocyte agglutination and (b~ inhibition of erythrocyte agglutinatlon due to presence of analyte or antigen.
Fig 4. is a schematic representation depicting mechanisms of agglutination/non-agglutination in connection with an overlapping antigen assay.
DETAILED DESCRIPTION OF THE INVENTION
, In the agglutination assay of this invention, a reagent is provided which comprises an erythrocyte binding portion provided by an erythrocyte binding molecule attached to an analyte binding portion provided by an analyte binding molecule, or to an analyte analogue, without substantially changing the binding characterlstics of the binding portions. The reagent is non-agglutinating when incubated with endogenous erythrocytes in the absence of the analyte.
Ervthrocvte Bindinu Molecules Erythrocyte membranes contain various antigenic surface constituents, including proteins, glycoproteins, glycolipids and lipoproteins.
Antibodies which recognize these constituents may be preDared by conventional techniques using the membrane, or the purified constituents thereof, as immunogens. These antibodies may be monoclonal or polyclonal in nature. Either the intact.antibody, or specific binding fragments thereof, may be used as erythrocyte binding molecules (EBM). The antibody or antibody fragment may be polyvalent, divalent or univalent.
In addition, glycoproteins, glycolipids and other carbohydrate TLH/148c ~ 9 ~
1 30~652 structures on the surface of erythrocytes are recognized by chemicals kno~n as lectins, which have an affinity for carbohydrates. These lectins may also be used as EBMs. Other receptor molecules with specific affinity for the erythrocyte surface also may be used. These could also include molecules with an affinity for the lipid bilayer of the membrane. Examples of such molecules are: protamine, the membrane binding portion of the bee venom, melittin, and other very basic peptides.
The preferred EBMs of the present invention will recognize erythrocyte membrane constituents found on all, or nearly all erythrocytes, so that erythrocytes endogenous to the blood sample may be used as the agglutinating particles. Such constituents include the so-called "public antigens".
Erythrocyte membranes are lipid bilayers with a variety oF proteins either on the surface or with a hydrophobic portion a;lowing the protein to anchor ;n or pass through the membrane, and may have part of the molecule inside the cell.
Glycophorin A is an example of a molecule which traverses the cell membrane. The blood group specificity is conferred by carbohydrate or glycolipid moieties, which are attached to membrane proteins. It is thus ' important that an EBM should recognize either the protein part of a membrane glycoprotein constituent, which is common to all erythrocytes of a particular species or another common structure. The abi'lity of a bivalent EBM to agglutinate red cells will depend on steric factors, such as the mobility of the molecule and the position of the binding site above the lipid bilayer.
The proteins of erythrocyte membranes include:- glycophorin A (MN, Ena, Wrb), glycophorln B (Ss, 'N', U) and the minor constltuents, integral membrane protein 1 (Rhesus), membrane attached glycoprotein C4 TLH/148c - 10 -1 30~652 (Ch1do & Rodgers)~ lntegral membranQ glycopro~ein (an~on channel), glycolipids (L~-is), glycosphlngolipids ~AB~, li, P, ~k~, an~yr~n, spectrln, protein 4-1, F-actin. [The associated blood gro~p factols are in parentheses.]
The followlng publlcat~ons are incorpora~ed by reference:
1. Th~ red cell membrane. S.B. Shohet & E, Beutler, in: Hematology, 3rd ed. Fds: Williams, Beutler, Erslev & Lichtman, 1983.
(Revlew of all erythrocyte membrane antlgens)
TECHNICAL FIELD
.
The present invention relates to a reagent and a method for detecting an antigen, antibody or other analyte in human or animal blood by erythrocyte agglutination. The invention also concerr,s a kit containing the reagent and processes of preparation of the reagents.
BACKGROUND ART
Assaying blood samples for a particular antigen or antibody has traditionally involved the step of separating the cellular components from the serum components of the blood by centrifugation and/or clotting, prior to assay.
This presents several potentlal problems. Firstly, such an assay is not suited to testing being conducted under field conditions. In many veterinary situations a quick test in the field i5 more desirable than the alternative of transporting samples to laboratories for separation and assay. Also, veterinary surgeons who do not have access to a centrifuge frequently need to assay blood samples for the presence of infectious agents such as heartworm. Further, assays being used for the detection of diseases in Third World countries present a siiuation where simplicity and low cost are of the essence.
Secondly, in certain pathologic conditions, separation of the blood samples becomes difficult. Blood taken from patients suffering conditions such as Waldenstrom's macroglobulinemia is difficult to separate into serum ancl cell fractions making an assay which can be conducted on whole blood highly desirable.
Thirdly, blood samples are often used for testing tor the presence of highly contagious and potentially dangerous disease states. In these cases it is preferable that as little handling and processing of the samples as ~k TLH/148c - 1 -possible is undertaken in order to minimize the risk to personnel conducting the assay. Further, certain conditions make the provision of over-the-counter finger-prick assays highly desirable. Such assays must necessarily be suited to performance on whole blood.
Immunoassays have revolutionized human diagnostic and veterinary medicine since the introduction of techniques such as the radioimmunoassay, first reported by Yalow and Berson (1959) Nature 18~, 1648, and the enzyme immunoassay or EIA which was first reported by Engvall and Perlman (1971 Immunochem 8, 871 and Van ~eeman and Schuurs (1971) FEBS Letters 15, 232.
Whilst such assays are based on antibody-antigen interactions the detection systems utilized are usually complex. The reagents used are generally enzyme or radiolabelled antigens, antibodies or complexes thereof which require either incubation with specific substrates and measurement of a color end-point either visually or by means of a colorimeter or measurement of radioactive decay with radiation counters to detect the presence of the analyte being tested. These assays also in~olve several washing steps. Most immunoassays for the detection of analytes in blood are currently of this nature. Thus, whilst these assays are sensitive, they are lengthy and involve procedures which may require expensive instrumentation, for detection of the analyte under test.
An alternative to these assays is provided by immunoassays of the A type described by Gupta, et al., (1985) Journal of Immunological Methods 177-187. These are immunoassays in which erythrocytes and anti-erythrocyte antibodies are used in the indicator system. In these assays exogenous erythrocytes such as sheep erythrocytes are used.
In recent years it has been possible to attach antibodies tO latex beads, thus providing a rapid agglutination assay. This, however, still entails the separation of the serum/plasma phase from the cellular phase TLH/148c - ~ -and consequently requires the use of a centrifuge or filtration system.
Latex agglutination assays are described in Castelan, et al., J. Clin.
Pathol. (1968), 21, 638; and Singer & Plotz AM. J. Med.
~1956 (Dec)~, 888.
Both direct and indirect agglutination immunoassays are well known in the art. In these assays, the agglutination of particles to which antigen or antibody is bound is used to indicate the presence or absence of the corresponding antibody or antigen. A variety of particles, including particles of latex, charcoal, kaolinite, or bentonite, as well as both microbial and red blood cells, have been used as agglutinatable carriers.
See Mochida, US 4,308,026. The use of erythrocytes as indicator particles is strongly criticised by Patel, U5 3,882,225, who says that it is difficult to standardize indlcator erythrocyte 5.
Molinaro, US 4,130,634 describes an assay for an antigen which employs antibody-cc~ated red blood cells. Molinaro emphasizes that the method used to couple the antibody to the erythrocyte must not destroy the reactivity of the antibody. He makes it clear that antibodies which are specific for the erythrocyte are not useful for his assay. He does mention, however, the posslbility of using a hybrid antibody with one binding site specific for the antigen and the other specific for the red blood cell.
Chang, US 4,4~3,05~ discloses an agglutination immunoassay reagent in which two antibodies are covalently linked "ta~l-to-tail", i.e., so as not to alter their specificity. One antibody is specific for an antigen borne by an indicator substance, such as an erythrocyte. This antibody i5 preferably un~valent to avoid nonspecific agylutination. The other antibody is divalent and is speclflc for the analyte. In preparation for the assay, fresh erythrocytes are coated with the conjugate. The double TLh/148c - 3 -1 30~652 antibody conjugate-coated RBCs are then incubated ~Jith the test serum.
Chang does not contemplate the assaying of whole blood samples using a non-autoagglutinating anti-RBC antibody and endogenous erythrocytes.
Chu, US 4,493,793 discloses the construction of a lectin-antibody or lectin-antigen covalently coupled conjugate. His Table I (incorporated by reference) sets forth the carbohydrate specificities of several lectins.
He does not teach coupling such a conjugate to an erythrocyte through either the lectin or the antibody receptor.
Other "tail-to-tail" immunological conjugates are known. Segal, US
4,676,980 sets forth the construction of a "tail-to-tail" conjugate of a target cell surface antigen-specific antibody and of a cytotox~c effector cell receptor-specific antibody. Several cross-linking methods, incorporated by reference, are described. This conjugate is intended for use in immunotherapy, in that it will cause the cellular immune system of the patient to lyse the target cell. The target cell would not, of course, be an erythrocyte endogenous to the host.
Li, US 4,661,444 suggests the production of a tail-to-tail conjugate of an analyte-binding antibody and of an antibody specific for the idiotype of the first antibody. This conjugate was to be used in conjunction with an insolubilized analyte-binding antibody in an immunoassay.
~ ardlaw, US 4,695,553 teaches use of a monoclonal antibody against a universal erythrocyte antigen as a RBC ag~lutinating agent to clarify the interface between red blood cells and white blood cells in centrifuged whole blood. ~le prefers use of antibodies against glycophorin or against antigen, but also mentions the posslbility of using a mixture of lectins.
Guesdon, US 4,668,637 discusses the use of anti-red blood cell antibodies or of lectins for the purpose of erythroadsorption. Bigbee, [Molecular Immunology, ~0: 1353-1362 (1983)] describes the production and testing of TLH/148c - 4 -four monoclonal antibodies against glycophorin A. The general concept of using in an immunoassay an antibody which reacts with an antigenic determinant shared among all members of a class of analytes of interest (microorganisms) is set forth in McLaughlin, US 4,683,196.
A number of patents deal with antibodies useful in blood typing.
See, e.g., Lloyd, US 4,678,747; Graham, Jr., US 4,358,436; Liu, US
4,550,017; Steplewski, US 4,607,009; Lennox, W083103477. Tnese antibodies are useful for blood typing because they bind to antigens found only in certain blood cell populations, while for the purpose of this invention, it is desirable to use antibodies (or mixtures thereof) which bind to essentially all erythrocytes.
Zuk, US 4,594,327 recognizes the desirability of performing an immunoassay directly on whole blood samples. In his method, the sample is contacted with both an insolubilized, analyte-specific immunoreagent and with a red blood cell binding agent such as a RBC-specific antibody or a lectin. The analyte-specific immunoreagent and the ReC binding agent are not coupled together, and the assay disclosed is not an agglutination assay.
The problem, in an agglutination immunoassay, of nonspecific agglutination of erythrocytes by anti-erythrocyte antibodies endogenous to the blood sample, was noted by Czismas, US 3,639,558. He proposed eliminating all naturally occurring antigenic sites on the particle by coating the particle with protein.
Theofilopoulos, US 4,342,566; Duermeyer, US 4,292,403 and Goldenberg, US 4,331,6q7 are of interest as demonstrating the use of specific binding fragments of antibodies as substitutes for intact antibodies in assays.
The construction of heterobi~unctional antibodies is taught by Auditore-Hargreaves, US 4,446,233; Paulus, US 4,444,878; and Reading, US
4,474,893. Mochida, US 4,200,436 discloses the use of monovalent TLH/148c ~ 5 ~
antibodies or binding fragments thereof in certaln lmmunoa~says Forrest, US 4,65~B78 mentions that monovalent antlbodies cannot form dlmers or more extens1ve complexes with thq ant~en; such aggreg~tes w~re said to be c~pable of interfer1ng with ~he bind~ng of the antlgen-antibody complex to a solid phase support.
According to a flrst embodiment of thls lnvention there is prov~ded an agglutinatlon reagent for dlrect detcction of an analyte ln a blood sample, compris~ng an erythrocyte bindln~ molQcule attach2d to an analyte bindin~ molecule, whereln the erythrocyte bindlng molecule is attached to the analyte b~nd1ng molecule in such a manner that the blndlng ch~ractertstics of the ~tnding molecules are not altered by the attachment, and the erythrocyte blnding molecule 1s capable of bind~ng erythrocytes ~ndogenous to the blood sample but does not agglutina~e endogenous erythrocytes in the absence of analyte, wtth the proviso that when sald ery~hrocyte blndlng m~lecule ls an anti-erythrocyte-ant~body or part thereof, s~d analyte blnding mvlecule ls no~ an ant~body attached thereto by means of a he~srobl~u~ctlon~l couplln~ r~agent.
Acco~d~ng to a s~cond embodiment of this lnvention ther~ ls pr~vlded an agglutlnatlon reagent whlch co~pris~s a conJuga~e comprislng at least one erythrocyte bindtng molecule con~ugatcd with at least on~
~nal.yte b~nding molecule, sa~d cc,n~ugate agglutinatlng erythrocy~s essentlally only ln the presence o~ the analyte~ whereln sald con~ugate does not substantially alter the bindtng characteristlcs of satd erythrocyte bindlng molecule and sald analyte binding molecule or lyse said erythrocytes; and wherein satd erythrocyte ~nd~ng mol0cule ~s a i~tl-ulllvdlulll duLl-~lythl~cyto ~ntlbo~y, ~hereln ~Ql~ ~nttbody or fragment ~ss~nt~ally does no~ aut~-a~lut1n~te erythrocytes, or a ."~ .
fragment of such ~n ~ntibody .
According to a third embodlment of th~s In~ent10n there ls prov~ded an agglutlnatlon reagent for assaying analyte whlch comprlses a heterobifunctional antibody or a heterobl~unct~onal b~nd~n~ fra~ment of an ant~bo~y, said antibody consistl ng of an erythrocyt~ ~lnd~ng molecule which ~lnds erythrocytes but not the analyte, and an analyte blndln~
molecule b1nd~ng the analyte but not erythrocytes, the erythrocyte blndlng molecule belng con~ugatcd to the analyte blnding molecule by one or more dlsulphldQ bonds and not by a heteroblfunctlonal eoupllng agent.
Accordlng to a fourth e~bodiment of this invent10n there 1s provided an agglutlnation reagent whlCh comprlses a coniugate comprls~ng an erythrocyte b~ndlng molecule con~ugat~d w1th an analyte blndlng m~lecule whereln the erythrocyte blndlng molecule is a peptide hav~ng an a~finity for the erythrocyte membrane but lncapable Gf lyslng erythrocytes, and ls not derived from an antlbody or leetln~
Accordlng to a ~l~th embodlment of this in~ention there lS proY1ded a d1rect agglut~nation assay for the presenee of an analyte ln a blood ~ampl~ contain~ng erythrocytes whgch assay comprlses mlxlng the sample wl~h an agglutlnation reag~nt accordlng to any of the ~lrst to fourth embod~ents and obsQrvlng whether the erythr~cytes are a~glutlnated and correlatln~ the ag~tutinat~on w7th tne amount of analyte present 1n ~he sampl e .
Accordlng to a slxth embodiment of thls lnventlon there is provlded a dlrec~ agglutlna~lon assay ~or the presence or amoun~ of an analyte ln a sample whlch comprlses forming a mlxturQ of a sample, erythrocytQs~ and an agglutlnatlon reagent whlch reagent comprises a con~ugate ~ompris~ng an erythrocyt~ bindlng molecule con~ug2ted wlth an analyte b~ndlng molecule, wh~re1n th~ erythrocyte b~ndln~ mo~ecule is a p~ptlde h~ving an aff~n~ty for ~he erythrocyte membrane but incapable of lysinig erythrocyt~s, and ls not d~rlved from an ant~body or l~ctln; ~bservln~
wheth~r the erythrocytes are agglutlnatQd and dlrectly correlat~ng the presence or amount oP agglutlnatlon wlth the presence or amount o~
analyte present.
Accordlng to a seventh ~mbodlment o~ thls lnventlon there ls provided a d~rect agglutlnation assay for the presence of an analyte ln a whole blood çample from a sub~ec~ which comprises contRctlng a blood samp7e containtng erythrocytes endogenous to the sub~Qct w1th an agglutination reagent whlch compris~s a conjugate of (a) a multivalent monoclonal antlbody which blnds to ~rythrocyte membranes or a b~nd1n~
~ragment thereof, and (b) an analyte blndlng molecule, sald con~ugate belng essentl~lly lncapable of agglutinatlng sald erythrocytes ln the abs~nce of analyte, observing wheth~r th~ ery~hrocytes are ag~lutlnated and dlrectly correlat1n~ the ~gglutlnatlon w~th the presence or amount o~
analyte present.
Accordlng to an e~ghth e~o~lment of th1s lnventlon thQre ls prov~d~d an agglutlnatlon ass~y for an analyte laeklng repeat~ng epitope~
wh~ch compr1s~s ~ncubattng a sample whlch i~ay contaln such analyte, erythrocytes, a con~ugate o~ an erythrocyte blnding molecule and an analyte b1ndlng molecule, and a non-unlva~ent secondary b~ndlng molecul~
whlch binds to a new epltope formed by the blnding of the analyte blndlng molecule to the analyte, and correlat1ng the presence or d~gree of agglutlnation wlth the presence or quantlty o~ the analyte ln the sample.
Accordlng to a nlnth embodiment o~ this inventlon there ls provl~ed an agglutlnatlon assay ~or an analyte lackln~ repeat1ng epl~opes whlch comprises lncubatlng a sample which may contain such analyte, erythrocytes~ a con~ugate of an erythrocyte bindlng molecule and an ,, ~ .
,r .
analyte blnding molecule, where sa~d con~ugate essentlally does not auto-agglutinate-erythrocytes, and a secondary btndlng molecule which binds to a new epitope formed by thQ blnding o-f the analyte btndlng mo~ecule to tne analyte, and correlatlng the presence or d~gree of agglutinatlon wlth the presence or quantity of the analyte tn the sample, wheretn the secondary bindlng molecule ls also conju~ated to an erythrocyte blndSng molecule.
Accordlng to a tenth embodimen~ of thls tnv~ntlon there ls provlded a dlrect ag~lutlnation assay for the presence or amount of an analyte ~n a sample whlch comprtses forming a m~xture of a sample, erythrocytes7 and an agglutlnatlon reagent whlch comprlses a conju~ate comprls7ng an erythrocyte bind~ng molecule con~ugated wlth an analyt~ btndln~ molecule whereln the con~u~ate ls a heterobtfunc~10nal antlbody or a he~erobifuncttonal blnd1ng fra~ment o~ an antibody, said antibody conslsttn~ of an erythrocyte bindtn~ ant1body fragmQnt whlch blnds erythrocytes but not the an~lyte, and an analyte binding anttbvdy fra~ment blnding th~ analyte but not erythrocytes, the ~rythro~yte binding frag~ent b~ing conjugated to the analyt~ b1ndlng fl~g~Qnt by one or mDre dtsulphlde bonds and not hy a heterobtfunct~onal coupling agent, and wh~reln the reagen~ co~prlses a detectabl~ amount of homo~lfuncttonal erythrocyte-blndlng ant{body, but sald ho~oblfunctlonal erythrocyte-blndlng ant~body essentially does not auto-agglu~lnat~
erythrocytes.
Accordlng to an eleventh Qmbod~ment of thls invention there ~s prov1ded an agglutinat~on reagent for ~ndtrect de~ectton of an analyte ln a blood sample, c~mprlslng an erythrocyte blndlng molecule attached to an analyte analo~ue wherein satd erythroeyte binding molecule ts a~tached to salct analyte analogue in such a mannQr that blnding characterlsttcs o~
. .
the erythrocyte blnding molecule are not altered by the attachment, and said erythro~yt~ blndlng molecule is capable of binding erythrocytss endogenous to the sample but does not agglutlnate endogenous erythrocytes 1n the absence of an analyte blndlng reagent.
According to a twel~th embodlment of this invention ther@ 15 provided an agglutlnatlon reagent which comprlses a con~ugate c~mprls~ng an erythrocyte blndlng molecule conjugated wlth an analyte analogue where1n sa1d con~ugate does not substantlally alter the bindlng characteristlcs of the erythrocyte b~nding molecule (EBM), or the analyte analogue and does not lyse erythrocytes, wherein sald conjugate essent~ally does not agglutlnate erythro~ytes ln the absenc~ of an analyte binding rQagent~ and wherein sald EBM ls a non-unlYalent anti-ery~hrocyte ant1body or a ~ra~ment of such an antlbody whereln sald ant1~0~y or fragment essentlally do not auto-a~glutinate erythrocytes.
Accord~ng to a twelfth embodim~nt o~ th~s lnventlon there 1s provlded an agglutlnatlon reagent which comprlses a son~ugate comprising an erythrocyt~ bind1ng molecule conjug~d w1th an analyte analogu~, whQreln the erythrocyte b1nding ~olecule ls a p~ptlde havlng an aff1nlty for the erythrocyte membrane ~ut ~ncap~bte o~ lys1ng erythrocytes, and ts not ~er1vQd from an ant~body or lectin.
Accor~lng to a thirteenth embodiment of th~s ~nvont10n there ts provided an 1nd1rec~ agglut1natlon assay for the presence or amount of an analyte in a blood sample whlch comprises ~ncubatlng the sample w~th an agglut~na~lon reagent accordlng to the eteventh or twelfth embod~ment and a soluble ana1yte blnding reagent, whereby said agglutinatlon reag~nt compQte~ wlth sample analyt~ for the analyte blnding sltes of sa1d analyte blndlng reagent observing whether a~glutinat~on occurs, and determinlng the presence or am~unt of sa1d analyte from the lnverse o~
,~ ' the degree of ag~lut~nation.
Accordlng to~ fourteenth embodlmen~ of th~s invent~on there ls prov1ded an ind1rect agglutlnation assay for the presence or amount of the analyte in a sample which comprlses ~a) formln~ a ~xture of sample, erythrocytes, an agglutlnatlon reagent whSch comprises a con~u~ate compristng an erythrocyte binding molecule conjugated w~th an analyte binding molecule, wherein the erythrocyte b1ndln~ molecule is a peptlde having an a~finity for the erythrocyte membrane but lncapable of lysing ery~hrocytes, and 1s not derived from an antlbody or lect1n, and a soluble non-unival~nt analyte b~nding rQagent which is essentially ~ncapable on ~ts own of agglutlnating erythrocytes, (b) permttt~ng sa~d conjugate to compete with sampl~ analyte for the analyt~ ~lnd~ng sites o~
the analyte b1nd~ng reagen~, ~c) observing whether agglut1nation o~curs, and ~d) inversely correlating the degree of agglutinatlon w~th the amount of analyte pres~nt.
Accord~ng to a fl~tQenth embodlment of this invent~on there ls provided an ln~lrect ag~lutlnation assay for the presence o~ an analyte ~n a whole bl~od sample from a s~b~ect which comprises (a) contactln~ a blood sample containing erythrocytes endogenous to the sub~ect w1th Sl) an agglutlnation rea~ont wh~ch com~r~s~s a conjugate of an ~ntact multlvalent monoclonal ant1~ody wh~ch binds to erythrocyte me~ranes or a ~inding fragment thereo~, and an analyte analogue, satd con~ugate belng eapable of agglutinat1ng erythrocytes but only ln the presence of a mul~ivalent analyte blnding agent, and (iiS a soluble analyte blndln~
reagent wh~eh ~s essen~ially 1ncapable of agglu~inating erythrocytes, (b) permitting sa1d conjugat~ to compete with sample analyte for the analyte b~nding sites o~ ~he analytq b1nding rea~ent, ~c) observlng whether aggl~tlnatlon occurs, and ~dS inversely correlat~ng the degree o~
1 30~652 agglutlnat10n w1th the presence or amount of analyte present.
According to a sixteenth embodiment of thls inventlon there 1s p~ovlded a test kit for use 1n dlrect ag~lutlnatlon assays which compr~ses (a~ a con~u~ate of an ery~hrocyte b1nd~ng ~olecule and an analyte blnd1ng molecule ~al d con~ ugate be 1 ng capable of agglutlnating erythrocyte~ only ln the presence of the analyte and (b) a seeondary btndlng mole~ule whlch b~nds to a new epitope ~ormed by the blnd~ng of the analyte bindlng molecul~ to the analyte.
According to a seventeenth e~bod1ment of this 1nvention there ls provlded a dlrect agglu~lnation assay test k1t comprts~ng an agglutlnation reagent according to any one of the first to fourth embodiments and a reference solut~on contalning a known quantity of analyte.
Accord~ng to an eighteenth embodiment of ~hls inven~on there ~s prsvided a t~st kit for use ~n 1ndlrect ~gglut~nat10n assays which comprlses an aggluttn~tlon reagent according to the elevQnth or ~wel~h embod1men~ and a soluble analyte blndlng reagent.
The present tnventors reco~n~zed that there was a nced ~or a me~hod whtch can be us~d in the laboratory and 1n th~ f1eld part1cularly ln Thlrd World Countr~es where there is l~ck of medical testin~ ~ciltties for analysls of d1~ferent typ~s of ana1ytes in whole blood. As ~ndlca~2d above earli~r meth~ds requlre scpar~tlon of the blood cells from serum or plasma and are ther2fore d1fflcult and in many cases ~mposslble to ~mplement ~n the fleld.
If erythrocyte-bindlng molecules are coupled to sp2clflc analyte~
b1ndin~ mol~cules then the r~sulting con~ugate could be used to blnd both Ln53Qçn9u5 erythrocytes and analytes present 1n a blood sample.
The present tnventlon resul~s from the flndlng that when such a complex ... - 6F -, ~ ' 1 30~652 ls exposed to a blood sample, agglutlnatl~n of the erythrocytes endogenous to tha~.sample will serve as an Indicator of the pres~nce of the retevant analyte (usually, an antigen or antibody) due to cross-l~nklng of erythr~cytes wlth the analyte.
Adv~ntages of endogenous RBCs O ~
- no need to centrlfuge sample whole blo~d, collected in the presence o~ a suitab1e anticoagulant, is used instead of seru~
or pla~a.
- for samples ~rom patients with in~ectious diseases, such as AIDS or hepatit~s, there ~s mlnimal sample handling.
-/.
/
-, . ..
1 30~652 appropriate for mass screening programs as conducted by the ~orld Health Organization in third world countries, whose facilities are limited.
the assay is very robust; there is only a single reagent, which is stable in the presence of a bacteriostatic agent such as 0.01% (w/v) sodium azide.
can be used as a field test by veterinary practitioners, when the appropriate animal red cel1s are used for immunization to produce species specific MAb.
the test is very fast - agglutination occurs in less than three minutes.
the method can be used to monitor therapeutic drugs and patient compliance.
it also has possible use as an "over-the-counter" self testing assay.
the only equipment needed is a mixin~ stirk, glass or plastic slide, lancet and possibly a microcapillary.
Advantaqes over exoqenous ervthrocytes include:
no pretreatment of erythrocytes. Patent 4,433,059 issued February 21, 1984 to Chi-Deu Chang and ~enry A. Graham uses blood group O
negative cells, which have been spun down, reacted with antibody conjugate for 15-30 minutes and washed 3x in PBS. Patent 4,668,637 issued May 26, 1987 to Jean-Lue Guesdon and Stratis Avrameas uses sheep red blood cells, which have been washed and resuspended in PBS. After the reaction, which takes place on a solid support, the cells are then fixed.
no pretreatment of samples. Patent 4,433,059 (identified a~ove) notes that samples have to be heat inactivated to avoid interference due to complement. Rabbit serum and bovine albumin must also be added to minimize other non-specific reactions. None of this is 148c , .
.
necessary with the present systern, where undiluted whole blood from patients may be reacted directly with reagent. This reagent contains unrelated monoclonal antibody to prevent any anti-mouse reactions, which may occur.
Thus, it is possible to dispense with the cumbersome separation of cells from serum and with the sensitization and fixing of exogenous erythrocytes intended for use as indicator particles in agglutination assays. The endogenous erythrocytes are sensitized by the reagent.
Another novel aspect of applicants' agglutination reagents and assays is the selection of an erythrocyte binding molecule such that incubation of conjugate with endogenous erythrocytes will not cause agglutination of the erythrocytes unless analyte is also present -- such erythrocyte binding molecules are termed herein "non-auto-agglutinating~.
The erythrocyte binding molecule is preferably a monoclonal antibody and especially, in the human system, an anti-glycophorin antibody. It is believed that this antibody is non-autoagglutinating for steric reasons;
either the binding sites of the intact antibody are able only to bind adjacent epitopes on the same erythrocyte or only one of the two binding sites can bind to glycophorin at one time.
Applicant's assay can detect small antigens without repeatlng determinants, using two conjugates, one bearing an analyte-specific binding molecule and the other, a binding molecule specific for a new epitope formed by the binding of the first conjugate to the analyte. This allows cross-linking in the presence of the antigen to be measured.
Brief Description of the Drawinqs Fig. 1 is a schematic representation of erythrocytes showing positive and negative agglutination results with antibody complexes in the presence and absence of antigen respectively.
TLH/148c - 8 -``` 1 308652 Fig 2. is a schematic representation of erythrocytes showing positive and negative agglutination results with a complex of antibody and an antigen in the presence and absence, respectively of anti-antigen antibodies.
Fig 3. is a schematic representation depicting (a) erythrocyte agglutination and (b~ inhibition of erythrocyte agglutinatlon due to presence of analyte or antigen.
Fig 4. is a schematic representation depicting mechanisms of agglutination/non-agglutination in connection with an overlapping antigen assay.
DETAILED DESCRIPTION OF THE INVENTION
, In the agglutination assay of this invention, a reagent is provided which comprises an erythrocyte binding portion provided by an erythrocyte binding molecule attached to an analyte binding portion provided by an analyte binding molecule, or to an analyte analogue, without substantially changing the binding characterlstics of the binding portions. The reagent is non-agglutinating when incubated with endogenous erythrocytes in the absence of the analyte.
Ervthrocvte Bindinu Molecules Erythrocyte membranes contain various antigenic surface constituents, including proteins, glycoproteins, glycolipids and lipoproteins.
Antibodies which recognize these constituents may be preDared by conventional techniques using the membrane, or the purified constituents thereof, as immunogens. These antibodies may be monoclonal or polyclonal in nature. Either the intact.antibody, or specific binding fragments thereof, may be used as erythrocyte binding molecules (EBM). The antibody or antibody fragment may be polyvalent, divalent or univalent.
In addition, glycoproteins, glycolipids and other carbohydrate TLH/148c ~ 9 ~
1 30~652 structures on the surface of erythrocytes are recognized by chemicals kno~n as lectins, which have an affinity for carbohydrates. These lectins may also be used as EBMs. Other receptor molecules with specific affinity for the erythrocyte surface also may be used. These could also include molecules with an affinity for the lipid bilayer of the membrane. Examples of such molecules are: protamine, the membrane binding portion of the bee venom, melittin, and other very basic peptides.
The preferred EBMs of the present invention will recognize erythrocyte membrane constituents found on all, or nearly all erythrocytes, so that erythrocytes endogenous to the blood sample may be used as the agglutinating particles. Such constituents include the so-called "public antigens".
Erythrocyte membranes are lipid bilayers with a variety oF proteins either on the surface or with a hydrophobic portion a;lowing the protein to anchor ;n or pass through the membrane, and may have part of the molecule inside the cell.
Glycophorin A is an example of a molecule which traverses the cell membrane. The blood group specificity is conferred by carbohydrate or glycolipid moieties, which are attached to membrane proteins. It is thus ' important that an EBM should recognize either the protein part of a membrane glycoprotein constituent, which is common to all erythrocytes of a particular species or another common structure. The abi'lity of a bivalent EBM to agglutinate red cells will depend on steric factors, such as the mobility of the molecule and the position of the binding site above the lipid bilayer.
The proteins of erythrocyte membranes include:- glycophorin A (MN, Ena, Wrb), glycophorln B (Ss, 'N', U) and the minor constltuents, integral membrane protein 1 (Rhesus), membrane attached glycoprotein C4 TLH/148c - 10 -1 30~652 (Ch1do & Rodgers)~ lntegral membranQ glycopro~ein (an~on channel), glycolipids (L~-is), glycosphlngolipids ~AB~, li, P, ~k~, an~yr~n, spectrln, protein 4-1, F-actin. [The associated blood gro~p factols are in parentheses.]
The followlng publlcat~ons are incorpora~ed by reference:
1. Th~ red cell membrane. S.B. Shohet & E, Beutler, in: Hematology, 3rd ed. Fds: Williams, Beutler, Erslev & Lichtman, 1983.
(Revlew of all erythrocyte membrane antlgens)
2. The red eell membrane skeleton. V.T. Marchesi. 8100d, 1~83. ~Revtew of the skeleton proteins~
An especially preferred EBM 1s one recognlzlng glycophortn, When erythrocyte staloglycopeptides ~re extracted from membranes, the main fraction (approximately 75~ of to~al) ~s glycophorin. Th~s molecule comprlses 131 am~no acids w~th 16 oligosaccharlde cha~ns. ~hus7 ~his ~s an abundant mo12ty, whlch could allow antlbody attachment wlthout agglutinating the red cells. It 1s also read11y avallable 1n a relatively purQ forlP co~merc1ally, e.g., from Sigma Che~lcal Company.
(See "Fra~tlon~tion of the Ma~or S1alo01ycopept1des o~ the human Red Blood Cell Membran~ H. Furthmayr, M. Tomlta ~ V.T. Marches1. 8~R~
1975, 113-122).
~ hen the erythrocyte b1nd~ng molecule !s multivalent, as ln the case of a normal antlbody, 1~ is des1rable ~hat the molecule reco~nize an erythrocyte membrane const1tuent whlch is abundant and well-dlstributed, and the bindlng site snould b~ ln s~ch a posltlon that crosslinking between cells ls lnh1bit0d by ster~c hindranfe, thereby avolding premature red cell agglutlnatlon.
Alternat1vely, cross-11nking may be inhiblt~d by the selection of an EBM that recogn1?es a surface constltuent present in sufflclen~
A~
~
quantity so that the epitopes are sufflciently close for the binding sites on the EB~-to be bound by one RBC.
It ls preferable, bu~ not necessary, that a single EBM be used that recognize~ essent7ally all erythrocytes. Several EBMs may be used, elther ln ~he sa~e or in separate eon~ugates, ~ach of wh~ch r0cognizes a particular group of erythrocytes, but wh1ch in aggregate recogni2e essentlally ~11 erythrocytes.
Whlle lt ~s preferable that the EBM recogn~ze a natural surface constituent of the erythrocyte, it ls poss1ble to coat erythrocytes with a ligand re~ognlzed by the EBM, or to treat the erythrocytes so as to expose a normally cryptic ligand.
Th1s inventlon ls not lim7ted to the detect70n of ~ny partlcular ana)yte. Th~ analy~e may be a substance normally found in blood, such as a blood protein or a hormone, or i~ may be a foreign substance, such as drug (includlng both therapeutSc drugs and drugs of abuse~, or an or~anism, such as a v1rus (by recognl~1ng a virus coat protein) bacterium, proto~oan, fungus, or multicellular parasite ~e.g., he~rtworm).
The analy~e may h~ve ~peatin~ ~p~topes, recogn7zable by one analyte blnding molecule, or ~n1que epitopes, where a mlxture of analytQ
b7nd1ng molecules ~s necessary. However, analytes whlch can only be bound by one ABM at a time, may ~lso be detected.
~n3~ ~e Bindinq Molecule The analyte blndlng molecule ~ay be any substance hav7ng a preferen~lal a~flnlty for the analyte, 1nclud~ng monoclonal or polyclonal antibodies lect1ns, ~n~ymes, or other blnding proteins or substances (or b~nd1ng fragments thereof). ~here tnQ ~nalyte ls an an~gen, the AB~ ls usually an antlbody. Wh~re ~he analy~e is an an~body, th~ A~M ~s .~ .
1 30~652 usual ly a~ antigen recognized by that antibody. When the analyte to be detected has n~ repeatlng ~p~topes, ~wo or more ABMs are requ~red wlth dlf-ferent speci~lcities for the analyte. The ~eagent in ~his case will be elther a m~xture of EBM bound to ABMl and EBM bound to A~M2, or EBM
w~th both ABMs a~tached.
The analyte bindlng molecule need not blnd the analyte directly.
~Llinq o~ ~X_lQd ~BM
The EBM and the ABM may be coupled together dlrectly or indirectly, and by covalent or non-covalent means ~or a comb1nation thereof~. ~here multiple EBMs or ABMs are used> EBMs or A8Ms may ~e coupled together, wlth one or more ABMs coupt~d directly to an EBM. The followin~ table surnmarizes some of the covalent co~pllng methods known in the art.
~ergblfunct~Qn~l 1. SPDP
- ~N-Succ1n~midyl~3,2-(pyridyldithio)proplona~e) ~io~r4~h~ , 7 \~ ~1 , M~h, . MaS
~m-male~ldobenzoyl-N-hydroxysucc1nimlde esterS
K~tagaw, ~_al.. 1976, 3. ~oche~., 79, 223-~36.
An especially preferred EBM 1s one recognlzlng glycophortn, When erythrocyte staloglycopeptides ~re extracted from membranes, the main fraction (approximately 75~ of to~al) ~s glycophorin. Th~s molecule comprlses 131 am~no acids w~th 16 oligosaccharlde cha~ns. ~hus7 ~his ~s an abundant mo12ty, whlch could allow antlbody attachment wlthout agglutinating the red cells. It 1s also read11y avallable 1n a relatively purQ forlP co~merc1ally, e.g., from Sigma Che~lcal Company.
(See "Fra~tlon~tion of the Ma~or S1alo01ycopept1des o~ the human Red Blood Cell Membran~ H. Furthmayr, M. Tomlta ~ V.T. Marches1. 8~R~
1975, 113-122).
~ hen the erythrocyte b1nd~ng molecule !s multivalent, as ln the case of a normal antlbody, 1~ is des1rable ~hat the molecule reco~nize an erythrocyte membrane const1tuent whlch is abundant and well-dlstributed, and the bindlng site snould b~ ln s~ch a posltlon that crosslinking between cells ls lnh1bit0d by ster~c hindranfe, thereby avolding premature red cell agglutlnatlon.
Alternat1vely, cross-11nking may be inhiblt~d by the selection of an EBM that recogn1?es a surface constltuent present in sufflclen~
A~
~
quantity so that the epitopes are sufflciently close for the binding sites on the EB~-to be bound by one RBC.
It ls preferable, bu~ not necessary, that a single EBM be used that recognize~ essent7ally all erythrocytes. Several EBMs may be used, elther ln ~he sa~e or in separate eon~ugates, ~ach of wh~ch r0cognizes a particular group of erythrocytes, but wh1ch in aggregate recogni2e essentlally ~11 erythrocytes.
Whlle lt ~s preferable that the EBM recogn~ze a natural surface constituent of the erythrocyte, it ls poss1ble to coat erythrocytes with a ligand re~ognlzed by the EBM, or to treat the erythrocytes so as to expose a normally cryptic ligand.
Th1s inventlon ls not lim7ted to the detect70n of ~ny partlcular ana)yte. Th~ analy~e may be a substance normally found in blood, such as a blood protein or a hormone, or i~ may be a foreign substance, such as drug (includlng both therapeutSc drugs and drugs of abuse~, or an or~anism, such as a v1rus (by recognl~1ng a virus coat protein) bacterium, proto~oan, fungus, or multicellular parasite ~e.g., he~rtworm).
The analy~e may h~ve ~peatin~ ~p~topes, recogn7zable by one analyte blnding molecule, or ~n1que epitopes, where a mlxture of analytQ
b7nd1ng molecules ~s necessary. However, analytes whlch can only be bound by one ABM at a time, may ~lso be detected.
~n3~ ~e Bindinq Molecule The analyte blndlng molecule ~ay be any substance hav7ng a preferen~lal a~flnlty for the analyte, 1nclud~ng monoclonal or polyclonal antibodies lect1ns, ~n~ymes, or other blnding proteins or substances (or b~nd1ng fragments thereof). ~here tnQ ~nalyte ls an an~gen, the AB~ ls usually an antlbody. Wh~re ~he analy~e is an an~body, th~ A~M ~s .~ .
1 30~652 usual ly a~ antigen recognized by that antibody. When the analyte to be detected has n~ repeatlng ~p~topes, ~wo or more ABMs are requ~red wlth dlf-ferent speci~lcities for the analyte. The ~eagent in ~his case will be elther a m~xture of EBM bound to ABMl and EBM bound to A~M2, or EBM
w~th both ABMs a~tached.
The analyte bindlng molecule need not blnd the analyte directly.
~Llinq o~ ~X_lQd ~BM
The EBM and the ABM may be coupled together dlrectly or indirectly, and by covalent or non-covalent means ~or a comb1nation thereof~. ~here multiple EBMs or ABMs are used> EBMs or A8Ms may ~e coupled together, wlth one or more ABMs coupt~d directly to an EBM. The followin~ table surnmarizes some of the covalent co~pllng methods known in the art.
~ergblfunct~Qn~l 1. SPDP
- ~N-Succ1n~midyl~3,2-(pyridyldithio)proplona~e) ~io~r4~h~ , 7 \~ ~1 , M~h, . MaS
~m-male~ldobenzoyl-N-hydroxysucc1nimlde esterS
K~tagaw, ~_al.. 1976, 3. ~oche~., 79, 223-~36.
3. SIAB
~N-succin~mldyl~q~lodoacetylamlnobenzo~) ~elt~an, ~ al., 1983., Bio. Techniques, 1. 1~8-152.
Sele~tive ~ifunctiQDal P-lsothlocyanatoben20ylchloride US Patent 4 6~0 338 ~i functl Q~alL
1. 3SOCOES
. ~
BisC2-(succlnlmidooxycarbonyloxy)ethyl]sulphone ?arllng,~ al,, 1980, J~ Xmmunol., 124, 913-g20 f'~ .
2. BS
Bis~sulphosuccinimidyl)suberate Staros, 1982, Biochemistry, 21, 3950-3955.
Other 1. Glutaraldehyde Avrameas, 1969, Immunochem., 6, 43.
2. Periodate Oxidation Nakane and Kawoi, 1974, J. Histochem. Cytochem., 22, 1084-1091.
3. Carbodiimide Of the foregoing methods of covalent coupling, conjugation with SPDP
is preferred.
The EBM and the ABM may also be coupled noncovalently, for example, by (a) attaching biotin to one and avidin (or strepavidin) to the other), (b) attaching an anti-antibody to one, which then binds the other, (c) attaching Protein A to one, which then binds the Fc portion of the other, and (d) attaching a sugar to one and a corresponding lectin to the other.
It should be understood that in coupling the EBM and the ABM, the binding characteristics should be changed as little as possible. It may be advantageous to provide a spacer moiety between the EBM and the ABM to reduce steric hindrance.
The EBM/ABM conjugate may be a chimeric antibody. One method of constructing such a conjugate is the following:
(a) preparing F(ab)2 fragments of a selected antibody by pepsin digestion;
(b) reducing and treating the fragments with Ellman's reagent to produce Fab' fragments of the selected antibody;
(c) thiolysing a selected specific antibody or a selected erythrocyte antibody: and TLH/148c - 14 -(d~ coupling the thioylated Fab' fragment to the Ellman's reagent treated Fab' fragment to produce a chirneric anti-erythrocyte antibody-antigen specific antibody conjugate.
Another method is set forth below:
(a) treating an anti-erythrocyte monoclonal antibody-producing hybridoma and an antigen specific monoclonal antibody-producing hybridoma with a distinct site-specific irreversible inhibitor of macromolecular biosynthesis;
(b) fusing the two different monoclonal antibody-producing hybridomas with polyethylene glycol ~c) cloning the fused cells either in soft agarose or by limiting dilution;
(d) selecting cloned heterohybridomas secreting chimeric anti-erythrocyte antibody-antigen specific antibody with a screening assay appropriate to the antibodies, (e) purifying the antibody product by affinity purification to free it from non-hybrid antibcdies.
Preferably the inhibitor is selected from the group consisting of emetine, actinomycin D, hydroxyurea, ouabain, cycloheximide, edine and sparsomycin.
The chimeric ant~body may be two half-molecules, one with specificity for erythrocytes (the EBM) and the other with specificity for the analyte (the ABM). ln this case the disulfide bonds of the antibody couple the ABM
to the EBM to ~orm the conjugate. Alternatively, the two half-molecules may be specific for the same or different epitopes of the analyte. In this second case, the chimeric antibody is really two ABMs and must be coupled to an EBM to form a tripartite conjugate. Tripartite conjugates may be formed by other means, such as attaching the EBM and two ABMs to a macromolecular spacer.
The simplest agglutination reagent contemplated is one comprising a TLH/148c - 15 -I 30~652 single conjugate of one EBM to one ABM. This reagent is suitable for the detection of antigens with repeating epitopes.
Antigenic analytes large enough to allow simultaneous b~nding of two antibody molecules, but which lack repeating epitopes, are known. They include many peptide and protein hormones. For agglutination to occur, the antigen must interact with the reagent so that at least some ~olecules of antigen act as a bridge between proximate erythrocytes. For assaying such analytes, it is preferably to employ a reagent comprising two or more distinct conjugates, i.e., ABMl/EBM + ABM2/EBM where ABMl and ABM2 bind to different, non-overlapping epitopes of the analyte. One mlght instead use a more complex single conjugate, ABMl/ABM2/EBM, where the spacial conformation is unlikely to favor the binding of both ABMs on the same conjugate molecule to the sa~e analyte molecule.
ErythrocYte AgQlutination Assay Both direct and indirect agglutination assays are known in the art.
In the conventional direct assay for an antigen, red cells are coated with antibody, and reacted with the sample. Multifunctional antigens act as bridges between the coated red blood cells, creating an agglutinate. In the conventional indirect assay, red cells are coate~ with antigen, and contacted with both a soluble antibody and with sample. Sample antigen competitively inhibits the binding of the sensitized red cells by the antibody, and hence the agglutination. It is also possible to additionally use an antibody - sensitized RBC. See Mochida, U.S. 4,308,026.
The reagent of the present invention may be used in either a direct or an indirect agglutination assay format. However, unlike conventional assays, it is not necessary to preeoat erythrocytes with antibody or antigen. Rather, the reagent may be added to a blood sample containing endogenou; erythrocytes, whereupon it will sensitize the cells, rendering 1 TLH/148c - 16 -1 30~652 them able to bind sample an~lyte (a direct assay) or to compete with sample analyte for a soluble analyte-binding m~lecule (~n indirect assay).
For some small circulating molecules such as synthetic or natural steriods, i.e., digoxin, theophylline, etc., or drugs of abuse, i.e., phenobarbital, cannabinoids, opioids, etc., the analyte in question may be too small to provide the two necessary antigenic epitopes for antibody binding (or other "epitopes" for recognition by other binding molecules) to allow cross-linking and subsequent erythrocyte agglutination.
For the assay of small molecules, as in drug monitoring or indeed for any other antigens, an agglutination inhibition assay is preferred. In this case, a two stage test is expected. The first stage would be addi~ion of a reagent consisting of the analyte or analyte analogue coupled to the non-agglutinating EBM, and the second stage would be addition of an uncon~ugated ABM. (The two stages may be reversed). If analyte is present in ~he blood sample, the specific blnding of the ABM to the EBM-analyte analogue conjugate will be inhibited, leading to a loss of agglutination.
Otherwise, agglutination occurs.
The term "analyte analogue" includes both the analyte and any substance also specifically bound by the ABM when such binding is competively inhibited by the analyte. The analyte analogue may be anti-idiotypic antibody raised against the antigen-binding site of an analyte-binding antibody.
For the detection of such small molecules by direct agglutination assay, at least two specific monoclonal antibodies could be used. One monoclonal antibody which is capable of binding directly ~o the small circulating antigen would be coupled to the erythrocyte binding molecule.
The second (secondary) monoclonal antibody would be incubated with the TLUtl48c - 17 -``" 1 30~652 above conjugate and the analyte and would be capable of binding to a new antigenic determinant comprised of an overlapping region of the first monoclonal antibody and the antigen that exists only when the first monoclonal antibody binds antigen. Thus, the second monoclonal antibody acts as the erythrocyte "bridge", finally causes cross-linking between different red cells allowing agglutination to occur. This method, of course, is not restricted to monoepitopic analytes.
Because of spacial conformation, it may be difficult for a s mgle secondary-antibody molecule to bind simultaneously to two conjugate:
analyte complexes. Thus, it may be preferable to conjugate the secondary antibody with an erythrocyte binding molecule.
In stating that a sample is to he incubated with a plurality of reagents it is to be understood that the contact may be simultaneous or sequential, without limitation to any particular order or duration of contact.
EXAMPLE 1 - Pre~aration of erthy~ocvte binding molecule (ant -glvcoDhorin antibody) Immunization and Screening Procedure Mice were immunized with human red blood cells and monoclonal antibodies produced by fusing the spleen cells of immunized animals with mouse myeloma cells. The antibodies were screened by both spin agglutination assay and enzyme immunoassay, where glycophorin was bound to a microtitre plate. Spin agglutination was performed by a modification of Wyatt & Street, Aust. J. Med. Lab. Sci, 4 48-50. 50 ~1 of cell culture suDernatant was mixed with 50 ~1 of a lX red blood cell suspension in a microtitre plate. For this example, antibodies which bound glycophorin, but did not agglutinate, were selected. The reaction of monoc10nal antibody and glycophorin was determined by enzyme immunoassay. Microplates . ~ .
TLH/148c - 18 -1 30~652 were coated with 10 micrograms/ml human glycophorin [Sigma Cat. No. G 7389]
and washed, then incubated with serial dilutions of monoclonal antibody.
After further washing, the presence of bound antibody was determined by the addition of enzyme labelled anti-mouse antibodies followed by the addition of substrate. The titre was determined to the largest dilution of monoclonal, which gave an A420 reading greater than 0.1 OD units above background.
Of 384 wells, 40 primary cloner were chosen. These gave either a positive spin agglutination assay, a response to slycophorin on EIA or both.
EIA Spin a~qlutination Number of clones Nesative Positive 4 Positive Positive 20 Positive Negative 16 Subsequent absorption studies were performed to confirm that the antibodies recognized a glycophorin domain exposed on the red c~ll surface.
The results of the screening assays on ascitic fluid are listed below:
Ascitic Fluid Titre Spin Red Cell CloneAqqlutinationGlycophorin EIA Absorption Test RAT lD3/167512000 <1000 Positive RAT 3D615 6400 1024000 Positlve RAT lC3/86<1000 1024000 Positive RAT 3Bl/172256000 2000 Positive RAT 3D3/22 4000 1024000 Positive RAT 3D5/61128000 1024000 Positive RAT lA2/187<1000 256000 Positive RAT 2A2/187clooo 128000 Pcsitive RAT lA3/129~1000 12800 Weak RAT lC4/5 <1000 128000 Positive TLH/148c - i9 -1 30~652 RAT 4C3/13 <1000 128000 Positive RAT 3Blt70 <1000 517000 Positive RAT IC3t86 has been deposited under the Budapest Treaty, with the designation G 26.4.IC3/86, ATCC ~ 1 ~ at the American Type Culture Collection, 12301 Parklawn Drive, Rockville MD, 20852, USA.
Purification of RAT lC3/86 Monoclonal antibodies were purified to homogeneity from ascitic fluids by chromatography on hydroxylapatite (Stanker, et al., J. Immunol.
Methods 76, 157, 1985).
EXAMPLE 2: Preparation of HIV pePtide - Ab coniuqate The spread of the human immuno deficiency virus (HIV-l~ has become a major global health problem. At present there is no recognised cure or vaccine for this disease. The diagnosis of infected individuals is a major factor in attempts to curtail the spread of the virus. Moreover, the need to prevent blood product contamination and protect health care personnel has increased the demand for simple, rapid, inexpensive and specific tests for the presence of anti-HIV antibodies.
We have made use of the patient's own red cells to provide a potential detection system for anti-HIV antibodies. This has been accomplished by selecting a non-agglutinating monoclonal antibody to human red blood cells. Chemically cross-linking this antibody with a synthetic HIV peptide antigen permitted specific agglutination of patients' red cells in the presence of antibodies to this antigen. The synthetic peptide antigen derived from gp41 of HIV-l (residues 579-602), was chosen on the basis of the Welling procedure, FEBS LETT. 188: 215 (1985) and corresponds with the region identified as a major epitope recognised by antibodies from approximately 98Z of AIDS patients. ~Wang et al Proc. Nat. Acad. Sci. USA
83: 6259 (1986)].
TLH/148c - 20 -I 30~652 Synthetic peptides were synt~esized using the Merrifield procedure [RS Hodges and RB Merrifield, L~l Biochem. 65, 241 (1975)] with the aid of an Applied 6iosystem~s M,odel 430 synthesizer using double coupling cycles ~d~ T,~ r supplied by the ~rflt$~t~Yr. The N-t-butyloxycarbonyl amino acid derivatives were obtained from the Protein Research Foundation (Osaka, Japan). Side chain protection was the same as supplied by Applied Biosystems with the exception of arginine for which the omega-N02 derivative was used. Chain assembly was monitored using ninhydrin ~V Sarin et al Anal. Biochem. 117, 147 (1981)~. The assembled peptides were simultaneously cleaved and deprotected using anhydrous HF containing lOX
anisole (v/v) [JM Stewart and JD Young, Solid Phase Peptide Synthesis, pp44 and 66, WH Freeman, San Francisco (1966)]. The crude peptide was precipitated with diethylether, washed with ethylacetate, and extracted with 60Z acetonitrile in 0.1% trifluoroacetic acid (v/v). Synthetic peptides were purified by preparative reverse phase chromotography (Amicon C18 resin, 250A pore size 25 x 400 mm), eluting with a gradient of l,OOOml, O to 60% acetonitrile in 0.1% trifluoroacetic acid. The synthetic peptide was approximately 95% pure as judged by analytical reversed phase HPLC and by quantitative amino acid analysis following acid hydrolysis.
1. SPDP labellinq of the ervthrocvte binding Ab (RAT lC3/86) To 0.25 ml of 13.8 mg/ml RAT lC3/86 was added 12.5 ~1 of 2 mg/ml 5PDP in dimethyl formamide and the reaction was allowed to proceed for 1 hour at 25C. Unreacted SPDP was removed by gel filtration on Sephadex G
25 and the level of SPDP labelling (1.4 moles/mole) was determined.
2. Reduction of_peptide 3.2 Peptide 3.2 (sequence RILAVERYLKD~QLLGI~GCSGK, corresponding to residues 579-601 of the major coat protein of HIV 1) was dissolved in 1 ml of lOOmM Tris-M~L, lmM EDTA pH 8.0 and reacted with 10 ~1 of h'~ l TLH/148c - 21 -1 30~652 2-mercaptoethanol for 45 minutes at 40C. The reaction was terminated by the addition of 4 drops of trifluoroacetic acid~(TFA) and 1 ml of aqueous O.lZ. TFA. The mixture was applied to a Sep-pak (Waters) C 18 cartridge th~at had been treated with 20 ml of 60% acetonitrile)_ 4~ and equilibrated with 0.1% TFA. The reduced peptide was cycled through the Sep-pak twice before washing with 20 ml 0.1% TFA. The reduced peptide was eluted from the Sep-pak with 2 x 2 ml of 60% acetonitrile, 0.1% TFA. The sample was rotary evaporated to dryness prior to coupling.
3. Conjugation The peptide was dissolved in 0.2 ml of a buffer containing lOOmM
potassium phosphate, lOOmM sodium chloride and 4M guanidine HCl pH 7.4 and mixed with 2.2 mg of SPDP~labelled antibody in the same buffer, but without guanidine HCl. The flask was incubated overnight at 25C.
The degree of substitution of the antibody influenced the solubility of the conjugate; 20 moles peptide per mole of conjugate became insoluble.
The range 5-7 moles of peptide per mole antibody was optimal. The capacity of the conjugate to bind red blood cells was monitored using the agglutination test with rabbit antimouse antibody and with HIV positive whole blood.
~N-succin~mldyl~q~lodoacetylamlnobenzo~) ~elt~an, ~ al., 1983., Bio. Techniques, 1. 1~8-152.
Sele~tive ~ifunctiQDal P-lsothlocyanatoben20ylchloride US Patent 4 6~0 338 ~i functl Q~alL
1. 3SOCOES
. ~
BisC2-(succlnlmidooxycarbonyloxy)ethyl]sulphone ?arllng,~ al,, 1980, J~ Xmmunol., 124, 913-g20 f'~ .
2. BS
Bis~sulphosuccinimidyl)suberate Staros, 1982, Biochemistry, 21, 3950-3955.
Other 1. Glutaraldehyde Avrameas, 1969, Immunochem., 6, 43.
2. Periodate Oxidation Nakane and Kawoi, 1974, J. Histochem. Cytochem., 22, 1084-1091.
3. Carbodiimide Of the foregoing methods of covalent coupling, conjugation with SPDP
is preferred.
The EBM and the ABM may also be coupled noncovalently, for example, by (a) attaching biotin to one and avidin (or strepavidin) to the other), (b) attaching an anti-antibody to one, which then binds the other, (c) attaching Protein A to one, which then binds the Fc portion of the other, and (d) attaching a sugar to one and a corresponding lectin to the other.
It should be understood that in coupling the EBM and the ABM, the binding characteristics should be changed as little as possible. It may be advantageous to provide a spacer moiety between the EBM and the ABM to reduce steric hindrance.
The EBM/ABM conjugate may be a chimeric antibody. One method of constructing such a conjugate is the following:
(a) preparing F(ab)2 fragments of a selected antibody by pepsin digestion;
(b) reducing and treating the fragments with Ellman's reagent to produce Fab' fragments of the selected antibody;
(c) thiolysing a selected specific antibody or a selected erythrocyte antibody: and TLH/148c - 14 -(d~ coupling the thioylated Fab' fragment to the Ellman's reagent treated Fab' fragment to produce a chirneric anti-erythrocyte antibody-antigen specific antibody conjugate.
Another method is set forth below:
(a) treating an anti-erythrocyte monoclonal antibody-producing hybridoma and an antigen specific monoclonal antibody-producing hybridoma with a distinct site-specific irreversible inhibitor of macromolecular biosynthesis;
(b) fusing the two different monoclonal antibody-producing hybridomas with polyethylene glycol ~c) cloning the fused cells either in soft agarose or by limiting dilution;
(d) selecting cloned heterohybridomas secreting chimeric anti-erythrocyte antibody-antigen specific antibody with a screening assay appropriate to the antibodies, (e) purifying the antibody product by affinity purification to free it from non-hybrid antibcdies.
Preferably the inhibitor is selected from the group consisting of emetine, actinomycin D, hydroxyurea, ouabain, cycloheximide, edine and sparsomycin.
The chimeric ant~body may be two half-molecules, one with specificity for erythrocytes (the EBM) and the other with specificity for the analyte (the ABM). ln this case the disulfide bonds of the antibody couple the ABM
to the EBM to ~orm the conjugate. Alternatively, the two half-molecules may be specific for the same or different epitopes of the analyte. In this second case, the chimeric antibody is really two ABMs and must be coupled to an EBM to form a tripartite conjugate. Tripartite conjugates may be formed by other means, such as attaching the EBM and two ABMs to a macromolecular spacer.
The simplest agglutination reagent contemplated is one comprising a TLH/148c - 15 -I 30~652 single conjugate of one EBM to one ABM. This reagent is suitable for the detection of antigens with repeating epitopes.
Antigenic analytes large enough to allow simultaneous b~nding of two antibody molecules, but which lack repeating epitopes, are known. They include many peptide and protein hormones. For agglutination to occur, the antigen must interact with the reagent so that at least some ~olecules of antigen act as a bridge between proximate erythrocytes. For assaying such analytes, it is preferably to employ a reagent comprising two or more distinct conjugates, i.e., ABMl/EBM + ABM2/EBM where ABMl and ABM2 bind to different, non-overlapping epitopes of the analyte. One mlght instead use a more complex single conjugate, ABMl/ABM2/EBM, where the spacial conformation is unlikely to favor the binding of both ABMs on the same conjugate molecule to the sa~e analyte molecule.
ErythrocYte AgQlutination Assay Both direct and indirect agglutination assays are known in the art.
In the conventional direct assay for an antigen, red cells are coated with antibody, and reacted with the sample. Multifunctional antigens act as bridges between the coated red blood cells, creating an agglutinate. In the conventional indirect assay, red cells are coate~ with antigen, and contacted with both a soluble antibody and with sample. Sample antigen competitively inhibits the binding of the sensitized red cells by the antibody, and hence the agglutination. It is also possible to additionally use an antibody - sensitized RBC. See Mochida, U.S. 4,308,026.
The reagent of the present invention may be used in either a direct or an indirect agglutination assay format. However, unlike conventional assays, it is not necessary to preeoat erythrocytes with antibody or antigen. Rather, the reagent may be added to a blood sample containing endogenou; erythrocytes, whereupon it will sensitize the cells, rendering 1 TLH/148c - 16 -1 30~652 them able to bind sample an~lyte (a direct assay) or to compete with sample analyte for a soluble analyte-binding m~lecule (~n indirect assay).
For some small circulating molecules such as synthetic or natural steriods, i.e., digoxin, theophylline, etc., or drugs of abuse, i.e., phenobarbital, cannabinoids, opioids, etc., the analyte in question may be too small to provide the two necessary antigenic epitopes for antibody binding (or other "epitopes" for recognition by other binding molecules) to allow cross-linking and subsequent erythrocyte agglutination.
For the assay of small molecules, as in drug monitoring or indeed for any other antigens, an agglutination inhibition assay is preferred. In this case, a two stage test is expected. The first stage would be addi~ion of a reagent consisting of the analyte or analyte analogue coupled to the non-agglutinating EBM, and the second stage would be addition of an uncon~ugated ABM. (The two stages may be reversed). If analyte is present in ~he blood sample, the specific blnding of the ABM to the EBM-analyte analogue conjugate will be inhibited, leading to a loss of agglutination.
Otherwise, agglutination occurs.
The term "analyte analogue" includes both the analyte and any substance also specifically bound by the ABM when such binding is competively inhibited by the analyte. The analyte analogue may be anti-idiotypic antibody raised against the antigen-binding site of an analyte-binding antibody.
For the detection of such small molecules by direct agglutination assay, at least two specific monoclonal antibodies could be used. One monoclonal antibody which is capable of binding directly ~o the small circulating antigen would be coupled to the erythrocyte binding molecule.
The second (secondary) monoclonal antibody would be incubated with the TLUtl48c - 17 -``" 1 30~652 above conjugate and the analyte and would be capable of binding to a new antigenic determinant comprised of an overlapping region of the first monoclonal antibody and the antigen that exists only when the first monoclonal antibody binds antigen. Thus, the second monoclonal antibody acts as the erythrocyte "bridge", finally causes cross-linking between different red cells allowing agglutination to occur. This method, of course, is not restricted to monoepitopic analytes.
Because of spacial conformation, it may be difficult for a s mgle secondary-antibody molecule to bind simultaneously to two conjugate:
analyte complexes. Thus, it may be preferable to conjugate the secondary antibody with an erythrocyte binding molecule.
In stating that a sample is to he incubated with a plurality of reagents it is to be understood that the contact may be simultaneous or sequential, without limitation to any particular order or duration of contact.
EXAMPLE 1 - Pre~aration of erthy~ocvte binding molecule (ant -glvcoDhorin antibody) Immunization and Screening Procedure Mice were immunized with human red blood cells and monoclonal antibodies produced by fusing the spleen cells of immunized animals with mouse myeloma cells. The antibodies were screened by both spin agglutination assay and enzyme immunoassay, where glycophorin was bound to a microtitre plate. Spin agglutination was performed by a modification of Wyatt & Street, Aust. J. Med. Lab. Sci, 4 48-50. 50 ~1 of cell culture suDernatant was mixed with 50 ~1 of a lX red blood cell suspension in a microtitre plate. For this example, antibodies which bound glycophorin, but did not agglutinate, were selected. The reaction of monoc10nal antibody and glycophorin was determined by enzyme immunoassay. Microplates . ~ .
TLH/148c - 18 -1 30~652 were coated with 10 micrograms/ml human glycophorin [Sigma Cat. No. G 7389]
and washed, then incubated with serial dilutions of monoclonal antibody.
After further washing, the presence of bound antibody was determined by the addition of enzyme labelled anti-mouse antibodies followed by the addition of substrate. The titre was determined to the largest dilution of monoclonal, which gave an A420 reading greater than 0.1 OD units above background.
Of 384 wells, 40 primary cloner were chosen. These gave either a positive spin agglutination assay, a response to slycophorin on EIA or both.
EIA Spin a~qlutination Number of clones Nesative Positive 4 Positive Positive 20 Positive Negative 16 Subsequent absorption studies were performed to confirm that the antibodies recognized a glycophorin domain exposed on the red c~ll surface.
The results of the screening assays on ascitic fluid are listed below:
Ascitic Fluid Titre Spin Red Cell CloneAqqlutinationGlycophorin EIA Absorption Test RAT lD3/167512000 <1000 Positive RAT 3D615 6400 1024000 Positlve RAT lC3/86<1000 1024000 Positive RAT 3Bl/172256000 2000 Positive RAT 3D3/22 4000 1024000 Positive RAT 3D5/61128000 1024000 Positive RAT lA2/187<1000 256000 Positive RAT 2A2/187clooo 128000 Pcsitive RAT lA3/129~1000 12800 Weak RAT lC4/5 <1000 128000 Positive TLH/148c - i9 -1 30~652 RAT 4C3/13 <1000 128000 Positive RAT 3Blt70 <1000 517000 Positive RAT IC3t86 has been deposited under the Budapest Treaty, with the designation G 26.4.IC3/86, ATCC ~ 1 ~ at the American Type Culture Collection, 12301 Parklawn Drive, Rockville MD, 20852, USA.
Purification of RAT lC3/86 Monoclonal antibodies were purified to homogeneity from ascitic fluids by chromatography on hydroxylapatite (Stanker, et al., J. Immunol.
Methods 76, 157, 1985).
EXAMPLE 2: Preparation of HIV pePtide - Ab coniuqate The spread of the human immuno deficiency virus (HIV-l~ has become a major global health problem. At present there is no recognised cure or vaccine for this disease. The diagnosis of infected individuals is a major factor in attempts to curtail the spread of the virus. Moreover, the need to prevent blood product contamination and protect health care personnel has increased the demand for simple, rapid, inexpensive and specific tests for the presence of anti-HIV antibodies.
We have made use of the patient's own red cells to provide a potential detection system for anti-HIV antibodies. This has been accomplished by selecting a non-agglutinating monoclonal antibody to human red blood cells. Chemically cross-linking this antibody with a synthetic HIV peptide antigen permitted specific agglutination of patients' red cells in the presence of antibodies to this antigen. The synthetic peptide antigen derived from gp41 of HIV-l (residues 579-602), was chosen on the basis of the Welling procedure, FEBS LETT. 188: 215 (1985) and corresponds with the region identified as a major epitope recognised by antibodies from approximately 98Z of AIDS patients. ~Wang et al Proc. Nat. Acad. Sci. USA
83: 6259 (1986)].
TLH/148c - 20 -I 30~652 Synthetic peptides were synt~esized using the Merrifield procedure [RS Hodges and RB Merrifield, L~l Biochem. 65, 241 (1975)] with the aid of an Applied 6iosystem~s M,odel 430 synthesizer using double coupling cycles ~d~ T,~ r supplied by the ~rflt$~t~Yr. The N-t-butyloxycarbonyl amino acid derivatives were obtained from the Protein Research Foundation (Osaka, Japan). Side chain protection was the same as supplied by Applied Biosystems with the exception of arginine for which the omega-N02 derivative was used. Chain assembly was monitored using ninhydrin ~V Sarin et al Anal. Biochem. 117, 147 (1981)~. The assembled peptides were simultaneously cleaved and deprotected using anhydrous HF containing lOX
anisole (v/v) [JM Stewart and JD Young, Solid Phase Peptide Synthesis, pp44 and 66, WH Freeman, San Francisco (1966)]. The crude peptide was precipitated with diethylether, washed with ethylacetate, and extracted with 60Z acetonitrile in 0.1% trifluoroacetic acid (v/v). Synthetic peptides were purified by preparative reverse phase chromotography (Amicon C18 resin, 250A pore size 25 x 400 mm), eluting with a gradient of l,OOOml, O to 60% acetonitrile in 0.1% trifluoroacetic acid. The synthetic peptide was approximately 95% pure as judged by analytical reversed phase HPLC and by quantitative amino acid analysis following acid hydrolysis.
1. SPDP labellinq of the ervthrocvte binding Ab (RAT lC3/86) To 0.25 ml of 13.8 mg/ml RAT lC3/86 was added 12.5 ~1 of 2 mg/ml 5PDP in dimethyl formamide and the reaction was allowed to proceed for 1 hour at 25C. Unreacted SPDP was removed by gel filtration on Sephadex G
25 and the level of SPDP labelling (1.4 moles/mole) was determined.
2. Reduction of_peptide 3.2 Peptide 3.2 (sequence RILAVERYLKD~QLLGI~GCSGK, corresponding to residues 579-601 of the major coat protein of HIV 1) was dissolved in 1 ml of lOOmM Tris-M~L, lmM EDTA pH 8.0 and reacted with 10 ~1 of h'~ l TLH/148c - 21 -1 30~652 2-mercaptoethanol for 45 minutes at 40C. The reaction was terminated by the addition of 4 drops of trifluoroacetic acid~(TFA) and 1 ml of aqueous O.lZ. TFA. The mixture was applied to a Sep-pak (Waters) C 18 cartridge th~at had been treated with 20 ml of 60% acetonitrile)_ 4~ and equilibrated with 0.1% TFA. The reduced peptide was cycled through the Sep-pak twice before washing with 20 ml 0.1% TFA. The reduced peptide was eluted from the Sep-pak with 2 x 2 ml of 60% acetonitrile, 0.1% TFA. The sample was rotary evaporated to dryness prior to coupling.
3. Conjugation The peptide was dissolved in 0.2 ml of a buffer containing lOOmM
potassium phosphate, lOOmM sodium chloride and 4M guanidine HCl pH 7.4 and mixed with 2.2 mg of SPDP~labelled antibody in the same buffer, but without guanidine HCl. The flask was incubated overnight at 25C.
The degree of substitution of the antibody influenced the solubility of the conjugate; 20 moles peptide per mole of conjugate became insoluble.
The range 5-7 moles of peptide per mole antibody was optimal. The capacity of the conjugate to bind red blood cells was monitored using the agglutination test with rabbit antimouse antibody and with HIV positive whole blood.
4. Gel Filtration ChromatoqraphY
Unreacted peptide and SPDP by-prod,ucts ~ere removed by gel filtration Pha~
on a Superose~ 6 column (Pharmacia) in/~he~r~buffered saline and antibody containing fractions were pooled and stored at 4C after addition of 0.01 sodium azide as a preservative.
Unreacted peptide and SPDP by-prod,ucts ~ere removed by gel filtration Pha~
on a Superose~ 6 column (Pharmacia) in/~he~r~buffered saline and antibody containing fractions were pooled and stored at 4C after addition of 0.01 sodium azide as a preservative.
5. Preparation of reagent for assay Two volumes of conjugates were mixed with one volume of a 10 mg/ml solution of an unretated monoclonal antibody (Bruce 5) prepared as described in Bundesen, et al., Vet. Immun., Immunopath. 8, 245-260, 1985.
~ d~ J~kf TLH/148c - 22 -1 30~652 Assay procedure For assay, 10 ~1 of heparinized whole blood was placed on a glass slide. 30 yl of reagent was added and mixed. The slide was rocked for up to three minutes and the presence or absence of agglutination noted.
Nine independent peptide/antibody conjugates were prepared and found to be active in agglutinating seropositive patient's red blood cel'ls.
Active conjugate was also prepared using m-Maleimidobenzoy'1-N-hydroxysuccinimide ester as the cross-linking reagent.
Resul~s were at l~east comparable in accuracy to those observed with h~d~5 y an enzyme ~ 5t~t using a similar antigen (Table 1).
Comparative testing of blood samples was by means of ELISA for the purpose of confirming positives and negatives obtained with the erythrocyte assay.
Control blood samples comprised ELISA negative blood samples and ELISA positive samples from infected patients. HIV positive patients were confirmed western blot positive by the Victorian State Reference Laboratory. ~abiJf ~ld hospital patients were negative either by western blo,t or EIA (Abbe~Laboratories). Blood donors were tested by EIA ~Genetie Systems). False positive or negative values are given in parentheses and were verified by EIA or western blot analysis.
Autoloqous Red Cel1 Aq~ nation Test Agglutination EIA Test Test -~ve -ve +ve -ve HIV +ve patients 42 ~1) 43 0 TLH/148c - 23 -1 30~652 Fairfield Hospital patients (3) fi3 0 66 Healthy blood donors (1) 873 (2) 872 In order to evaluate the specificity of the test a series of synthetic peptides corresponding to other regions of the HIV-l envelope proteins was tested for their capacity to inhibit agglutination reaction (Table 2). No unrelated peptide competed and the synthetic gp41 fragment, residues 572-591, which is missing the essential carboxyterminal epitope region, did not inhibit agglutination. The inhibition of agglutination with free synthetic antigen was useful in confirming the occasional weak positive samples. If addition of synthetic peptide had failed to inhibit agglutination it would have been indicative of a false positive related to the anti-red blood cell antibody.
Synthetic peptide (0.125 mg/ml) was added to the conjugated antibody prior to the addition of whole blood. The agglutination test was performed as described above. Common sequences are underlined.
5cecifi_ ~tv of pe~t_d _ n ibition of aqqlutination Added Synthetic Inhibition of Peptide (sequence)Agglutination none OX
gp41 (579-601) RILAVERYLKDOOLLGIWGCSGK lOOX
gp41 (572-591) GIKQLARILAVERYLKADOO 0%
gpl20 (193-200) ASTTTNYT 0 TLH/148c ?4 _ 1 30~652 gpl20 (105-117) HEDIISL~DQSLK 0%
gpl20 ~101-118) VEQMHEDIISL~DOSLKP 0%
gpl20 ~105_129)Y129 HEDIISL~SQSLK AVKLTPLCVSY 0X
EXAMPLE 3 - Preparation of Chimeric Antibodies (anti-glycophorin/
anti-human D-dimer) and use in assay for D-dimer Monoclonal antibodies RAT lC3/86 (anti-human red blood cell) and DD-lC3/108 (anti-human D-dimer as described by Rylatt, et al., 1983, Thrombosis Res., 31, 767-778) were digested with pepsin essentially as described by Hackman, et al., 1981, Immunology, 15, 429-436, and purified by chromotrography on a TSK-3000 SW column. 2 mg RAT lC3/86 was digested for 45 minutes with 1% w/w pepsin in a buffer containing 0.1M acetic acid, 70mM sodium chloride pH 3.5. Meanwhile, 2 mg DD-lC3/108 was digested with 1% w/w pepsin for 2 hours in the same buffer. The reacti~ns were terminated by the addi;:ion of 1.5M Tris to raise the pH~8. T~e F(ab)2 fragments were purified by gel filtration chromatography on~TSK-3000 SW
column.
Reduction of the F(ab)2, and subsequent blocking of the Fab fragment, was carried out as described by Brennan, et al., 1985, Science 229~ 81-83. A 3 mg/ml F(ab)2 preparation was treated with lmM
mercaptoethylamine, in the presence of 10mM sodium arsenite, for 16 hours at 25C. The Fab fragments were stabilized by reaction with 5.5'-dithiobis (2-nitroben2Oic acid) (Ellman's reagent) for 3 hours at 25~C. The Fab fragment was then purified by gel filtration chromatography on a TSK-3000 S~ column.
The thiol form of DD-lC3/108 was regenerated by reaction with 10mM
mercaptoethylamine for 30 minutes at 25~C. Excess reagent was removed by TLH/148c - 25 -.
gel filtration chromatography on a TSK-3000 SW column. A mixture of the thiol DD-lC31108 and the Ellman's reagent treated RAT lC3/86 was incubated for 16 hours at 25C as described by Brennan, et al. Finally, the chimeric antibody was purified by further gel filtration chromatography on a TSK-3000 SW column.
Preparation of reaqent Two volumes of 0.1 mg/ml chimeric antibody was mixed with one volume of 7.5 mg/ml unrelated monoclonal antibody (Bruce 5).
Assay procedure For assay, 10 ~1 of heparinized whole blood was placed on a glass slide. 30 ~1 of reagent was added and mixed. The slide was rocked for three minutes and in the presence of D-dimer agglutination was observed.
EXAMPLE 4 - Pre~aration of SPDP-coniuqated Digoxin/Anti-~lycophorin antibody con~u~
Preparation of_Di~xin/Ab coniuqate 1) Preparation of periodate oxidized digoxin.
2 ml lOOmM sodium periodate was added slo~y, d,ropwise, to 40 mg of ~a~
A digoxin (Sigma), suspended in 2 ml 95% ~ and the reaction was allowed to continue for 31~ in at 37C. ~he reaction was stopped by the addition of 60 ~1 of-~ ethandiol. Finally, the Schiff's base intermediate was stabilized by the addition of 2 ml 40mM cystine ~30 min: 37C) and subsequent reaction with 1 ml of 15 mg/ml sodium borohydride (16h: 25C).
2) Reduction of cystine/digoxin conjugate.
3 ml of cystine/digoxin con~ugate was reduced by the addition of 40 ~1 mercaptoethanol (40 min: 37~C) and the product purified by chromatography on a Waters Sep-Pak C 18 cartridge as described for the reduction of peptide in Example 1. After rotary evaporation, the TLH/148c - 26 -1 30~3652 sample was reacted with SPDP labelled RAT lC3/86, which had been labelled with 5 propyldithiopyridine groupslantibody as described in Example 1 (16h: 25C). Finally, the digoxin/antibody conjugate was purified by gel filtration chromatography on Superose 12.
EXAMPLE 5 - Preparation of HIV pePtide/anti-qlycophorin- F(ab)2 con~ugate An alternative reagent for the detection of anti-HIV antibodies uses an F(ab)2 derivative of the anti-glycophorin antibody.
RAT lC3/86 (2 mg/ml in 70mM acetate lOOmM sodium chloride pH 3.5) was digested with 10 ~g/ml pepsin (Sigma P6887) for 40 min at 37C and the reaction terminated by the addition of 1/10 vol l.5M Tris base. After overnight dialysis into a buffer containing 5mM sodium phosphate pH 8.0, the antibody fragment was purified by ion-exchange chromatography on DEAE
cellulose on a 5-300mM gradient of sodium phosphate pH 8Ø
SPDP labelling of the Ftab)2 fragment, reduction of the peptide 3.2, conjugation of peptide 3.2 to F(ab)2 RAT lC3/86, purification of the peptide conjugate, preparation of reagent for assay and testing procedure were carried out as described ~or the whole antibody conjugate.
F(ab)2 conjugates of other erythrocyte or analyte-binding antibodies may similarly be prepared and may be coupled to molecules other than the HIV peptide.
EXAMPLE 6 - Preparation of HIV peptide/anti-qlycophorin Fab' coni~
Another alternative reagent employs a univalent Fab' fragment as the EBM.
F(ab)2 RAT lC3/86 in phosphate buffered saline was incubated with lOmM mercaptoethanol for lh at room temperature. Then 15mM ~odoacetamide was added and the reaction allowed to proceed for 15 min in the dark.
Finally, the reaction was terminated by the by dialysis into 100 vol of phosphate buffered saline.
TLH/148c - ~7 -SPDP labelling of the s-carboxymethylated Fab', reduction of the peptide 3.2, conju~ation of peptide 3.2 to the Fab'-TNB (thionitrobenzoyl) RAT fragment of RAT lC3/86, purification of the peptide conjugate, preparation of reagent for assay and testing procedure were carried out as described for the whole antibody conjugate.
EXAMPLE 7 = Pre~aration of Melittin as alternative EBM
A peptide from the bee venom, melittin (CULTTGLPALISWIKRKRQQ), was used as an alternative to the erythrocyte binding monoclonal antibody.
This peptide binds to the erythrocyte surface without lysing the cell (deGrado WF, Kezdy FJ, Kaiser ET. J Am Chem Soc 1981; 103; 679-81). The peptide was synthesised by the Merrifield procedure (Hodges, Merrifield.
Anal Biochem 1975; 65; 241).
One advantage of using melittin as the EBM is that it and a peptide-type ABM may be synthesized as a single unit.
EXAMPLE 8_- Use of Avidin-Biotin l _ a~e to couple EBM and ABM
The ABM and EBM need not be covalently coupled. One alternative is avidin-biotin linkage.
Preparation of B otin-labelled melittin The melittin peptide (10 mg) was reduced with mercaptoethanol as described for the 3.7 peptide in Example 1. After rotary evaporation, the sample was resuspended in 2 ml O.lM Tris-HCl, SmM EDTA pH 8.0 and reacted with 1 ml dimethylsulfoxide (DMSO), containing 4.3 mg N-iodoacetyl-N-biotinylhexylenediamine ~Pierce). This was allowed to react for 15 minutes at room temperature and ~he biotinylated derivative separated from byproducts on a Sephadex~G10 column.
Preparation of Avidin-labelled pepti_e 3.2 The 3.2 peptide is coupled to avidin in the same manner as it was coupled to antibody in Example 1.
10~ d~ n,~
TLH/148c - 28 -1 30~652 AssaY
Sub-agglutinating doses of the avidin-labelled peptide are added to the red cells.
Notes It will be understood that the avidinated and biotinylated molecules may be interchanged.
TLH/148c - 29 -
~ d~ J~kf TLH/148c - 22 -1 30~652 Assay procedure For assay, 10 ~1 of heparinized whole blood was placed on a glass slide. 30 yl of reagent was added and mixed. The slide was rocked for up to three minutes and the presence or absence of agglutination noted.
Nine independent peptide/antibody conjugates were prepared and found to be active in agglutinating seropositive patient's red blood cel'ls.
Active conjugate was also prepared using m-Maleimidobenzoy'1-N-hydroxysuccinimide ester as the cross-linking reagent.
Resul~s were at l~east comparable in accuracy to those observed with h~d~5 y an enzyme ~ 5t~t using a similar antigen (Table 1).
Comparative testing of blood samples was by means of ELISA for the purpose of confirming positives and negatives obtained with the erythrocyte assay.
Control blood samples comprised ELISA negative blood samples and ELISA positive samples from infected patients. HIV positive patients were confirmed western blot positive by the Victorian State Reference Laboratory. ~abiJf ~ld hospital patients were negative either by western blo,t or EIA (Abbe~Laboratories). Blood donors were tested by EIA ~Genetie Systems). False positive or negative values are given in parentheses and were verified by EIA or western blot analysis.
Autoloqous Red Cel1 Aq~ nation Test Agglutination EIA Test Test -~ve -ve +ve -ve HIV +ve patients 42 ~1) 43 0 TLH/148c - 23 -1 30~652 Fairfield Hospital patients (3) fi3 0 66 Healthy blood donors (1) 873 (2) 872 In order to evaluate the specificity of the test a series of synthetic peptides corresponding to other regions of the HIV-l envelope proteins was tested for their capacity to inhibit agglutination reaction (Table 2). No unrelated peptide competed and the synthetic gp41 fragment, residues 572-591, which is missing the essential carboxyterminal epitope region, did not inhibit agglutination. The inhibition of agglutination with free synthetic antigen was useful in confirming the occasional weak positive samples. If addition of synthetic peptide had failed to inhibit agglutination it would have been indicative of a false positive related to the anti-red blood cell antibody.
Synthetic peptide (0.125 mg/ml) was added to the conjugated antibody prior to the addition of whole blood. The agglutination test was performed as described above. Common sequences are underlined.
5cecifi_ ~tv of pe~t_d _ n ibition of aqqlutination Added Synthetic Inhibition of Peptide (sequence)Agglutination none OX
gp41 (579-601) RILAVERYLKDOOLLGIWGCSGK lOOX
gp41 (572-591) GIKQLARILAVERYLKADOO 0%
gpl20 (193-200) ASTTTNYT 0 TLH/148c ?4 _ 1 30~652 gpl20 (105-117) HEDIISL~DQSLK 0%
gpl20 ~101-118) VEQMHEDIISL~DOSLKP 0%
gpl20 ~105_129)Y129 HEDIISL~SQSLK AVKLTPLCVSY 0X
EXAMPLE 3 - Preparation of Chimeric Antibodies (anti-glycophorin/
anti-human D-dimer) and use in assay for D-dimer Monoclonal antibodies RAT lC3/86 (anti-human red blood cell) and DD-lC3/108 (anti-human D-dimer as described by Rylatt, et al., 1983, Thrombosis Res., 31, 767-778) were digested with pepsin essentially as described by Hackman, et al., 1981, Immunology, 15, 429-436, and purified by chromotrography on a TSK-3000 SW column. 2 mg RAT lC3/86 was digested for 45 minutes with 1% w/w pepsin in a buffer containing 0.1M acetic acid, 70mM sodium chloride pH 3.5. Meanwhile, 2 mg DD-lC3/108 was digested with 1% w/w pepsin for 2 hours in the same buffer. The reacti~ns were terminated by the addi;:ion of 1.5M Tris to raise the pH~8. T~e F(ab)2 fragments were purified by gel filtration chromatography on~TSK-3000 SW
column.
Reduction of the F(ab)2, and subsequent blocking of the Fab fragment, was carried out as described by Brennan, et al., 1985, Science 229~ 81-83. A 3 mg/ml F(ab)2 preparation was treated with lmM
mercaptoethylamine, in the presence of 10mM sodium arsenite, for 16 hours at 25C. The Fab fragments were stabilized by reaction with 5.5'-dithiobis (2-nitroben2Oic acid) (Ellman's reagent) for 3 hours at 25~C. The Fab fragment was then purified by gel filtration chromatography on a TSK-3000 S~ column.
The thiol form of DD-lC3/108 was regenerated by reaction with 10mM
mercaptoethylamine for 30 minutes at 25~C. Excess reagent was removed by TLH/148c - 25 -.
gel filtration chromatography on a TSK-3000 SW column. A mixture of the thiol DD-lC31108 and the Ellman's reagent treated RAT lC3/86 was incubated for 16 hours at 25C as described by Brennan, et al. Finally, the chimeric antibody was purified by further gel filtration chromatography on a TSK-3000 SW column.
Preparation of reaqent Two volumes of 0.1 mg/ml chimeric antibody was mixed with one volume of 7.5 mg/ml unrelated monoclonal antibody (Bruce 5).
Assay procedure For assay, 10 ~1 of heparinized whole blood was placed on a glass slide. 30 ~1 of reagent was added and mixed. The slide was rocked for three minutes and in the presence of D-dimer agglutination was observed.
EXAMPLE 4 - Pre~aration of SPDP-coniuqated Digoxin/Anti-~lycophorin antibody con~u~
Preparation of_Di~xin/Ab coniuqate 1) Preparation of periodate oxidized digoxin.
2 ml lOOmM sodium periodate was added slo~y, d,ropwise, to 40 mg of ~a~
A digoxin (Sigma), suspended in 2 ml 95% ~ and the reaction was allowed to continue for 31~ in at 37C. ~he reaction was stopped by the addition of 60 ~1 of-~ ethandiol. Finally, the Schiff's base intermediate was stabilized by the addition of 2 ml 40mM cystine ~30 min: 37C) and subsequent reaction with 1 ml of 15 mg/ml sodium borohydride (16h: 25C).
2) Reduction of cystine/digoxin conjugate.
3 ml of cystine/digoxin con~ugate was reduced by the addition of 40 ~1 mercaptoethanol (40 min: 37~C) and the product purified by chromatography on a Waters Sep-Pak C 18 cartridge as described for the reduction of peptide in Example 1. After rotary evaporation, the TLH/148c - 26 -1 30~3652 sample was reacted with SPDP labelled RAT lC3/86, which had been labelled with 5 propyldithiopyridine groupslantibody as described in Example 1 (16h: 25C). Finally, the digoxin/antibody conjugate was purified by gel filtration chromatography on Superose 12.
EXAMPLE 5 - Preparation of HIV pePtide/anti-qlycophorin- F(ab)2 con~ugate An alternative reagent for the detection of anti-HIV antibodies uses an F(ab)2 derivative of the anti-glycophorin antibody.
RAT lC3/86 (2 mg/ml in 70mM acetate lOOmM sodium chloride pH 3.5) was digested with 10 ~g/ml pepsin (Sigma P6887) for 40 min at 37C and the reaction terminated by the addition of 1/10 vol l.5M Tris base. After overnight dialysis into a buffer containing 5mM sodium phosphate pH 8.0, the antibody fragment was purified by ion-exchange chromatography on DEAE
cellulose on a 5-300mM gradient of sodium phosphate pH 8Ø
SPDP labelling of the Ftab)2 fragment, reduction of the peptide 3.2, conjugation of peptide 3.2 to F(ab)2 RAT lC3/86, purification of the peptide conjugate, preparation of reagent for assay and testing procedure were carried out as described ~or the whole antibody conjugate.
F(ab)2 conjugates of other erythrocyte or analyte-binding antibodies may similarly be prepared and may be coupled to molecules other than the HIV peptide.
EXAMPLE 6 - Preparation of HIV peptide/anti-qlycophorin Fab' coni~
Another alternative reagent employs a univalent Fab' fragment as the EBM.
F(ab)2 RAT lC3/86 in phosphate buffered saline was incubated with lOmM mercaptoethanol for lh at room temperature. Then 15mM ~odoacetamide was added and the reaction allowed to proceed for 15 min in the dark.
Finally, the reaction was terminated by the by dialysis into 100 vol of phosphate buffered saline.
TLH/148c - ~7 -SPDP labelling of the s-carboxymethylated Fab', reduction of the peptide 3.2, conju~ation of peptide 3.2 to the Fab'-TNB (thionitrobenzoyl) RAT fragment of RAT lC3/86, purification of the peptide conjugate, preparation of reagent for assay and testing procedure were carried out as described for the whole antibody conjugate.
EXAMPLE 7 = Pre~aration of Melittin as alternative EBM
A peptide from the bee venom, melittin (CULTTGLPALISWIKRKRQQ), was used as an alternative to the erythrocyte binding monoclonal antibody.
This peptide binds to the erythrocyte surface without lysing the cell (deGrado WF, Kezdy FJ, Kaiser ET. J Am Chem Soc 1981; 103; 679-81). The peptide was synthesised by the Merrifield procedure (Hodges, Merrifield.
Anal Biochem 1975; 65; 241).
One advantage of using melittin as the EBM is that it and a peptide-type ABM may be synthesized as a single unit.
EXAMPLE 8_- Use of Avidin-Biotin l _ a~e to couple EBM and ABM
The ABM and EBM need not be covalently coupled. One alternative is avidin-biotin linkage.
Preparation of B otin-labelled melittin The melittin peptide (10 mg) was reduced with mercaptoethanol as described for the 3.7 peptide in Example 1. After rotary evaporation, the sample was resuspended in 2 ml O.lM Tris-HCl, SmM EDTA pH 8.0 and reacted with 1 ml dimethylsulfoxide (DMSO), containing 4.3 mg N-iodoacetyl-N-biotinylhexylenediamine ~Pierce). This was allowed to react for 15 minutes at room temperature and ~he biotinylated derivative separated from byproducts on a Sephadex~G10 column.
Preparation of Avidin-labelled pepti_e 3.2 The 3.2 peptide is coupled to avidin in the same manner as it was coupled to antibody in Example 1.
10~ d~ n,~
TLH/148c - 28 -1 30~652 AssaY
Sub-agglutinating doses of the avidin-labelled peptide are added to the red cells.
Notes It will be understood that the avidinated and biotinylated molecules may be interchanged.
TLH/148c - 29 -
Claims (102)
1. An agglutination reagent for direct detection of an analyte in a blood sample, comprising an erythrocyte binding molecule attached to an analyte binding molecule, wherein the erythrocyte binding molecule is attached to the analyte binding molecule in such a manner that the binding characteristics of the binding molecules are not altered by the attachment, and the erythrocyte binding molecule is capable of binding erythrocytes endogenous to the blood sample but does not agglutinate endogenous erythrocytes in the absence of analyte, with the proviso that when said erythrocyte binding molecule is an anti-erythrocyte antibody or part thereof, said analyte binding molecule is not an antibody attached thereto by means of a heterobifunctional coupling reagent.
2. An agglutination reagant which comprises a conjugate comprising at least one erythrocyte binding molecule conjugated with at least one analyte binding molecule, said conjugate agglutinating erythrocytes essentially only in the presence of the analyte, wherein said conjugate does not substantially alter the binding characteristics of said erythrocyte binding molecule and said analyte binding molecule or lyse said erythrocytes; and wherein said erythrocyte binding molecule is a non-univalent anti erythrocyte antibody, wherein said antibody essentially does not auto-agglutinate erythrocytes, or a fragment of such an antibody.
3. The reagent of claim 2 in which the antibody fragment is non-univalent.
4. An agglutination reagent for assaying analyte which comprises a heterobifunctional antibody or a heterobifunctional binding fragment of an antibody, said antibody consisting of an erythrocyte binding molecule which binds erythrocytes but not the analyte, and an analyte binding molecule binding the analyte but not erythrocytes, the erythrocyte binding molecule being conjugated to the analyte binding molecule by one or more disulphide bonds and not by a heterobifunctional coupling agent.
5. The reagent of claim 4 further characterised in that the reagent is prepared by forming a heteobifunctional hybrid of a homobifunctonal erythrocyte-binding antibody and a homobifunctional analyte-binding antibody, said reagent comprising a detectable amount of homobifunctional erythrocyte-binding antibody, wherein said homobifunctional antibody does not auto-agglutinate erythrocytes.
6. The reagent of any one of claims 1 to 3 wherein said erythrocyte binding molecule is an anti-erythrocyte antibody.
7. The reagent of claim 2 or 3, wherein the reagent comprises a plurality of conjugates. each of which binds to a different epitope of the analyte.
8. The reagent of claim 1, wherein at least one of said binding molecules is an antibody or specific binding fragment thereof.
9. The reagent according to claim 1 or 2, wherein the antibody is produced by cell line G26.4.IC3/86 accorded ATCC Deposit No. H89893 or an F(ab), or Fab' fragment of said antibody.
10. The reagent according to claim 1, wherein the erythrocyte binding molecule corresponds to a fragment of a lectin, said fragment having only one erythrocyte binding site.
11. The reagent according to any one of claims 1 to 3, wherein the erythrocyte binding molecule is a peptide having an affinity for the erythrocyte membrane but is incapable of lysing erythrocytes.
12. The reagent of claim 1 or 2, wherein said erythrocyte binding molecule comprises a univalent fragment derived from an anti-erythrocyte antibody and said analyte binding molecule comprises a univalent-fragment derived from an anti-analyte antibody.
13. The reagent according to claim 1, wherein said erythrocyte binding molecule is an anti-erythrocyte antibody, or F(ab)2 or Fab' fragment thereof or melittin or a specific binding fragment thereof.
14. An agglutination reagent which comprises a conjugate comprising an erythrocyte binding molecule conjugated with an analyte binding molecule wherein the erythrocyte binding molecule is a peptide having an affinity for the erythrocyte membrane but incapable of lysing erythrocytes, and is not derived from an antibody or lectin.
15. The reagent according to claim 1 wherein the attachment is by means of covalent coupling.
16, The reagent according to any one of claims 1 - 5, 14, wherein the erythrocyte binding molecule but not the analyte binding molecule binds a surface protein or glycoprotein.
17. The reagent according to any one of claims 1 - 5 and 14, further comprising a secondary antibody or specific binding fragment thereof which specifically recognizes an overlapping epitope formed by the binding of said analyte binding molecule to said analyte.
18. The reagent according to any one of claims 1 - 5 and 14, further comprising a secondary antibody or specific binding fragment thereof which specifically recognizes an overlapping epitope formed by the binding of said analyte binding molecule to said analyte, wherein said secondary antibody is provided in unconjugated form.
19. The reagent according to any one of claims 1 - 5 and 14, further comprising a secondary antibody or specific binding fragment thereof which specifically recognizes an overlapping epitope formed by the binding of said analyte binding molecule to said analyte, wherein said secondary antibody is attached to an erythrocyte binding molecule.
20. The reagent of claim 3, wherein said erythrocyte binding molecule is an anti-glycophorin antibody or a specific binding fragment thereof.
21. The reagent of claim 14, wherein the peptide corresponds to a non-lytic, erythrocyte-binding, fragment of melittin which does not by itself agglutinate erythrocytes.
22. The reagent according to any one of claims 1 - 5, 14, wherein the analyte is D-dimer or crosslinked fibrin degradation products.
230 The reagent according to any one of claims 1 - 5, 14, wherein the analyte is an antibody to HIV.
24. The reagent according to any one of claims 1 - 3 14, wherein the analyte binding molecule is a HIV-1 peptide, or hepatitis virus antigen, digoxin or antibody F(ab)2 or Fab' fragment thereof raised against human D-dimer, or hepatitis virus or an anti-idiotypic antibody.
25. The reagent according to any one of claims 1 - 3, 14, wherein the analyte binding molecule is a HIV-1 peptide, or hepatitis virus antigen, digoxin or antibody F(ab)2 or Fab' fragment thereof raised against human D-dimer, or hepatitis virus or an anti-idiotypic antibody and wherein the HIV-1 peptide is gp 41 peptide.
26. The reagent of claim 5 wherein the erythrocyte binding molecule binds glycophorin.
27. The reagent of claim 14 in which the analyte binding molecule is a peptide or protein, and the erythrocyte binding molecule and analyte binding molecule are conjugated by a simple peptide bond.
28. The reagent of claim 5 which is a heterobifunctional F(ab)2.
29. The reagent of claim 5 in which the erythrocyte binding molecule binds a canine red blood cell antigen other than glycophorin.
30. The reagent of claim 5 in which the analyte-binding molecule binds an antigen associated with canine heart-worm.
31. The reagent of claim 5 in which the analyte-binding molecule binds human D-dimer.
32. A direct agglutination assay for the presence of an analyte in a blood sample containing erythrocytes which assay comprises mixing the sample with an agglutination reagent according to any one of claims 1, 2, 4 or 14, and observing whether the erythrocytes are agglutinated and correlating the agglutination with the amount of analyte present in the sample.
33. A direct agglutination assay for the presence or amount of an analyte in a whole blood sample from a subject which comprises forming a mixture of sample, erythrocytes, and an agglutination reagent according to claim 3; observing whether the erythrocytes are agglutinated and directly correlating the agglutination with the amount of analyte present.
34. A direct agglutination assay for the presence or amount of an analyte in a sample which comprises forming a mixture of a sample, erythrocytes, and an agglutination reagent which reagent comprises a conjugate comprising an erythrocyte binding molecule conjugated with an analyte binding molecule, wherein the erythrocyte binding molecule is a peptide having an affinity for the erthrocyte membrane but incapable of lysing erythrocytes, and is not derived from an antibody or lectin;
observing whether the erythrocytes are agglutinated and directly correlating the presence or amount of agglutination with the presence or amount of analyte present.
observing whether the erythrocytes are agglutinated and directly correlating the presence or amount of agglutination with the presence or amount of analyte present.
35. A direct agglutination assau for the presence of an analyte in a whole blood sample from a subject which comprises contacting a blood sample containing erythrocytes endogenous to the subject with an agglutination reagent which comprises a conjugate of (a) a multivalent monoclonal antibody which binds to erythrocyte membranes or a binding fragment thereof, and (b) an analyte binding molecule, said conjugate being essentially incapable of agglutinating said erythrocytes in the absence of analyte, observing whether the erythrocytes are agglutinated and directly correlating the agglutinating with the presence or amount of analyte present.
36. The assay of claim 35, wherein the binding fragment is non-univalent; said assay being further characterized in that at no time are the sample or the conjugate with erythrocytes exogenous to the subject.
37. An agglutination assay for an analyte lacking repeating epitopes which comprises incubating a sample which may contain such analyte, erythrocytes, a conjugate of an erythrocyte binding molecule and an analyte binding molecule, and a non-univalent secondary binding molecule which binds to a new epitope formed by the binding of the analyte binding molecule to the analyte, and correlating the presence or degree of agglutination with the presence or quantity of the analyte in the sample.
38. An agglutination assay for an analyte lacking repeating epitopes which comprises incubating a sample which may contain such analyte, erythrocytes, a conjugate of an erythrocyte binding molecule and an analyte binding molecule, where said conjugate essentially does not auto-agglutinate erythrocytes, and a secondary binding molecule which binds to a new epitope formed by the binding of the analyte binding molecule to the analyte, and correlating the presence or degree of agglutination with the presence or quantity of the analyte in the sample, wherein the secondary binding molecule is also conjugated to an erythrocyte binding molecule.
39. A direct agglutination assay for the presence or amount of an analyte in a sample which comprises forming a mixture of a sample, erythrocytes, and an agglutination reagent which comprises a conjugate comprising an erythrocyte binding molecule conjugated with an analyte binding molecule wherein the conjugate is a heterobifunctional antibody or a heterobifunctional binding fragment of an antibody, said antibody consisting of an erythrocyte binding antibody fragment which binds erythrocytes but not the analyte, and an analyte binding antibody fragment binding the analyte but not erythrocytes, the erythrocyte binding fragment being conjugated to the analyte binding fragment by one or more disulphide bonds and not by a heterobifunctional coupling agent, and wherein the reagent comprises a detachable amount of homobifunctional erythrocyte-binding antibody, but said homobifunctional erythrocyte-binding antibody essentially does not auto-agglutinate erythrocytes.
40. A direct agglutination assay for the presence of an analyte in a blood sample containing erythrocytes which assay comprises mixing the sample with an agglutination reagent according to any one of claims 1, 2, 4 or 14, and observing whether the erythrocytes are agglutinated and correlating the agglutination with the amount of analyte present in the sample, and wherein the analyte is an antigen or hapten and the analyte binding molecule is an antibody.
41. A direct agglutination assay for the presence of an analyte in a blood sample containing erythrocytes which assay comprises mixing the sample with an agglutination reagent according to any one of claims 1, 2, 4 or 14, and observing whether the erythrocytes are agglutinated and correlating the agglutination with the amount of analyte present in the sample, and wherein the analyte is an antibody and the analyte binding molecule is an antigen or hapten recognized by said antibody.
42. A direct agglutination assay for the presence of an analyte in a blood sample containing erythrocytes which assay comprises mixing the sample with an agglutination reagent according to any one of claims 1, 2, 4 or 14, and observing-whether the erythrocytes are agglutinated and correlating the agglutination with the amount of analyte present in the sample, and wherein the analyte is an antigen or hapten and the analyte binding molecule is an antibody, and wherein the analyte is an antibody and the analyte binding molecule is an anti-idiotypic antibody.
43. A direct agglutination assay for the presence of an analyte in a blood sample containing erythrocytes which assay comprises mixing the sample with an agglutination reagent according to any one of claims 1, 2, 4 or 14, and observing whether the erythrocytes are agglutinated and correlating the agglutination with the amount of analyte present in the sample, and wherein the analyte is an antigen or hapten and the analyte binding molecule is an antibody, further comprising adding a secondary antibody which binds to a new epitope formed by the binding of the analyte binding molecule to the analyte.
44. The assay of any one of claims 33 to 36, further comprising adding a secondary antibody which binds to a new epitope formed by the binding of the analyte binding molecule to the analyte.
45. A direct agglutination assay for the presence of an analyte in a blood sample containing erythrocytes which assay comprises mixing the sample with an agglutination reagent according to any one of claims 1, 2, 4 or 14, and observing whether the erythrocytes are agglutinated and correlating the agglutination with the amount of analyte present in the sample, and wherein the analyte is an antigen or hapten and the analyte binding molecule is an antibody, and wherein the analyte lacks repeating epitopes.
46. The assay of any one of claims 33 to 36, wherein the analyte lacks repeating epitopes.
47. The assay of claim 37 or 38, wherein the secondary binding molecule is also conjugated to an erythrocyte binding molecule.
48. The assay of claim 34 in which the analyte binding molecule is a peptide or protein, and the erythrocyte binding molecule and analyte binding molecule are conjugated by a simple peptide bond.
49. An agglutination reagent for indirect detection of an analyte in a blood sample, comprising an erythrocyte binding molecule attached to an analyte analogue wherein said erythrocyte binding molecule is attached to said analyte analogue in such a manner that binding characteristics of the erythrocyte binding molecule are not altered by the attachment, and said erythrocyte binding molecule is capable of binding erythrocytes endogenous to the sample but does not agglutinate endogenous erythrocytes in the absence of an analyte binding reagent.
50. An agglutination reagent which comprises a conjugate comprising an erythrocyte binding molecule conjugated with an analyte analogue wherein said conjugate does not substantially alter the binding characteristics of the erythrocyte binding molecule (EBM), or the analyte analogue and does not lyse erythrocytes, wherein said conjugate essentially does not agglutinate erythrocytes in the absence of an analyte binding reagent, and wherein said EBM is a non-univalent anti-erythrocyte antibody or a fragment of such an antibody wherein said antibody or fragment essentially do not auto-agglutinate erythrocytes.
51. The reagent of claim 50 in which the antibody fragment is non-univalent.
52. The reagent of claim 49, wherein the erythrocyte binding molecule is a peptide having an affinity for the erythrocyte membrane but is incapable of lysing erthrocytes.
53. The reagent of claim 49, wherein the erythrocyte binding molecule is a peptide having an affinity for the erythrocyte membrane, the analyte analogue is a peptide or protein, and said erythrocyte binding molecule and analyte analogue are conjugated by a simple peptide bond.
54. The reagent of claim 52, wherein the peptide is a nonlytic, erythrocyte binding fragment which does not by itself agglutinate erythrocytes.
55. An agglutination reagent which comprises a conjugate comprising an erythrocyte binding molecule conjugated with an analyte analogue, wherein the erythrocyte binding molecule is a peptide having an affinity for the erythrocyte membrane but incapable of lysing erythrocytes, and is not derived from an antibody or lectin.
56. The reagent according to any one of claims 49 to 55, wherein the analyte is an antibody to HIV.
57. The reagent of claim 49, wherein said erythrocyte binding molecule is an antibody or specific binding fragment thereof.
58. The reagent according to claim 57, wherein the antibody is produced by cell line G26.4.IC3/86 accorded ATCC Deposit No. HB9893 or an F(ab)2, or Fab' fragment of said antibody.
59. The reagent according to claim 49, wherein the erythrocyte binding molecule corresponds to a fragment of a lectin, said fragment having only one erythrocyte binding site.
60. The reagent of claim 49, wherein said erythrocyte binding molecule comprises a univalent fragment derived from an anti-erythrocyte antibody and said analyte binding molecule comprises a univalent fragment derived from an anti-analyte antibody.
61. The reagent according to claim 49 or 50, wherein said erythrocyte binding molecule is an anti-erythrocyte antibody, or F(ab)2 or Fab' fragment thereof or melittin or a specific binding fragment thereof.
62. The reagent according to claim 13, wherein the antibody or F(ab)2 or Fab' fragment thereof is raised against integral membrane protein 1, membrane attached glycoprotein C4, integral membrane glycoprotein, ankyrin, spectrin, glycophorin, glycolipid, glycosphingolipid, protein 4-1 or F-actin.
63. The reagent of claim 49, wherein the erythrocyte binding molecule is a peptide having an affinity for the erythrocyte membrane but incapable of lysing erythrocytes, and is not derived from an antibody or lectin.
64. The reagent according to any one of clalms 49 to 55, wherein the erythrocyte binding molecule but not the analyte binding molecule binds a surface protein or glycoprotein,
65. The reagent according to claim 49, wherein the attachment is by means of covalent coupling.
66. The reagent of any one of claims 1-5, 49-51, wherein the erythrocyte binding molecule is an antibody for glycophorin or a specific binding fragment thereof.
67. The reagent of any one of claims 1-57 49-51, wherein the erythrocyte binding molecule is an antibody for glycophorin or a specific binding fragment thereof, further characterized in that the erythrocyte binding molecule is an antibody for glycophorin A or a specific binding fragment thereof.
68. The reagent of claim 55, wherein the peptide is a nonlytic, erythrocyte-binding fragment which does not by itself agglutinate erytrocytes.
69. The reagent of claim 55 in which the analyte analogue is a peptide or protein, and the erythrocyte binding molecule and analyte analogue are conjugated by a simple peptide bond.
70. The reagent of any one of claims 14, 27, 49, 53, 55, 63 or 69, in which the erythrocyte binding molecule corresponds to a nonlytic, erythrocyte binding fragment of mellitin.
71. The reagent of any one of claims 21 or 68, in which the erythrocyte binding molecule corresponds to mellitin 7-26.
72. The reagent of claim 14 or 55 wherein the peptide corresponds to a nonlytic, erythrocyte-binding, fragment of protamine which does not by itself agglutinate erythrocytes.
73. An indirect agglutination assay for the presence or amount of an analyte in a blood sample which comprises incubating the sample with an agglutination reagent according to any one of claims 49, 50, 52 to 55 and a soluble analyte binding reagent, whereby said agglutination reagent competes with sample analyte for the analyte binding sites of said analyte binding reagent observing whether agglutination occurs, and determining the presence or amount of said analyte from the inverse of the degree of agglutination.
74. An indirect agglutination assay for the presence or amount of an analyte in a sample from a subject which comprises (a) forming a mixture of sample, erythrocytes and an agglutination reagent according to claim 51, (b) permitting said agglutination reagent to compete with sample analyte for the analyte binding sites of the analyte binding reagent, (c) observing whether agglutination occurs, and (d) inversely correlating the degree of agglutination with the presence or amount of analyte present.
75. An indirect agglutination assay for the presence or amount of the analyte in a sample which comprises (a) forming a mixture of sample, erythrocytes, an agglutination reagent which comprises a conjugate comprising an erythrocyte binding molecule conjugated with an analyte binding molecule, wherein the erythrocyte binding molecule is a peptide having an affinity for the erythrocyte membrane but incapable of lysing erythrocytes, and is not derived from an antibody or lectin, and a soluble non-univalent analyte binding reagent which is essentially incapable on its own of agglutinating erythrocytes, (b) permitting said conjugate to compete with sample analyte for the analyte binding sites of the analyte binding reagent, (c) observing whether agglutination occurs, and (d) inversely correlating the degree of agglutination with the amount of analyte present.
76. An indirect agglutination assay for the presence of an analyte in a whole blood sample from a subject which comprises (a) contacting a blood sample containing erythrocytes endogenous to the subject with (i) an agglutination reagent which comprise a conjugate of an intact multivalent monoclonal antibody which binds to erythrocyte membranes or a binding fragment thereof, and an analyte analogue, said conjugate being capable of agglutinating erythrocytes but only in the presence of a multivalent analyte binding agent, and (ii) a soluble analyte binding reagent which is essentially incapable of agglutinating erythrocytes, (b) permitting said conjugate to compete with sample analyte for the analyte binding sites of the analyte binding reagent, (c) observing whether agglutination occurs, and (d) inversely correlating the degree of agglutination with the presence or amount of analyte present.
77. The assay of claim 76, wherein the binding fragment is non-univalent; said assay being further characterized in that at no time are the sample or the conjugate with erythrocytes exogenous to the subject.
78. A direct agglutination assay for the presence of an analyte in a blood sample containing erythrocytes which assay comprises mixing the sample with an agglutination reagent according to any one of claims 1, 2, 4 or 14, and observing whether the erythrocytes are agglutinated and correlating the agglutination with the amount of analyte present in the sample, in which the sample is a whole blood sample and the sample and the conjugate are contacted essentially only with erythrocytes endogenous to the sample.
79. The assay of claim 33, in which the sample is a whole blood sample and the sample and the conjugate are contacted essentially only with erythrocytes endogenous to the sample.
80. An indirect agglutination assay for the presence or amount of an analyte in a blood sample which comprises incubating the sample with an agglutination reagent according to any one of claims 49, 50, 52 to 55 and a soluble analyte binding reagent, whereby said agglutination reagent competes with sample analyte for the analyte binding sites of said analyte binding reagent observing whether agglutination occurs, and determining the presence or amount of said analyte from the inverse of the degree of agglutination, in which the sample is a whole blood sample and the sample and the conjugate are contacted essentially only with erythrocytes endogenous to the sample.
81. The assay of any one of claims 74 to 77, in which the sample is a whole blood sample and the sample and the conjugate are contacted essentially only with erythrocytes endogenous to the sample.
82. The assay of claim 34 or 75 in which the peptide is a bee venom-like peptide.
83. The assay of claim 34 or 75 in which the erythrocyte binding molecule corresponds to a nonlytic, erythrocyte-biding fragment of melittin which does not by itself agglutinate erythrocytes.
84. The assay of claim 34 or 75 in which the erythrocyte binding molecule corresponds to a nonlytic, erythrocyte-biding fragment of melittin which does not by itself agglutinate erythrocytes in which the peptide corresponds to melittin 7-26.
85. The assay of claim 75 in which the analyte analogue is a peptide or protein and said erythrocyte binding molecule and analyte analogue are conjugated by a simple peptide bond.
86. The assay of claim 85 in which the erythrocyte binding molecule corresponds to a nonlytic, erythrocyte-binding fragment of melittin.
87. The assay of any one of claims 33, 74 or 77 in which the erythrocyte binding molecule is an anti-glycophorin monoclonal antibody or a specific binding fragment thereof.
88. The assay of any one of claims 33, 39, 74 or 77, wherein the erythrocyte-binding fragment is derived from an anti-glycophorin antibody.
89. The assay of claim 33 or 74, wherein the erythrocyte binding molecule is an antibody for a surface protein or glycoprotein or a specific binding fragment thereof.
90. The assay of claim 36, wherein the erythrocyte binding molecule is an antibody for glycophorin or a specific binding fragment thereof.
91. The assay of claim 33 or 74, wherein the erythrocyte binding molecule is an antibody for a surface protein or glycoprotein or a specific binding fragment thereof, wherein the erythrocyte binding molecule is an antibody for glycophorin or a specific binding fragment thereof.
92. The assay of claim 90, further characterized in that the erythrocyte binding molecule is an antibody for glycophorin A or a specific binding fragment thereof.
93. The assay of claim 33 or 74, wherein the erythrocyte binding molecule is an antibody for a surface protein or glycoprotein or a specific binding fragment thereof, wherein the erythrocyte binding molecule is an antibody for glycophorin or a specific binding fragment thereof, further characterized in that the erythrocyte binding molecule is an antibody for glycophorin A or a specific binding fragment thereof.
94. The assay of claim 76, wherein the agglutination reagent comprises a conjugate of (a) an intact multivalent monoclonal antibody which binds to erythrocyte membranes or a multivalent binding fragment thereof and (b) an analyte analogue, said conjugate being capable of agglutinating erythrocytes but only in the presence of a multivalent analyte binding agent.
95. A test kit for use in direct agglutination assays which comprises (a) a conjugate of an erythrocyte binding molecule and an analyte binding molecule, said conjugate being capable of agglutinating erythrocytes only in the presence of the analyte, and (b) a secondary binding molecule which binds to a new epitope formed by the binding of the analyte binding molecule to the analyte.
96. A direct agglutination assay test kit comprising an agglutination reagent according to any one of claims 1 to 5 and a reference solution containing a known quantity of analyte.
97. A test kit for use in direct agglutination assays which comprises an agglutination reagent according to any one of claims 1 to 5, and a secondary antibody which binds to a new epitope formed by the binding of the analyte binding molecule to the analyte.
98. The test kit of claim 95, wherein the secondary antibody is conjugated to an erythrocyte binding molecule.
99. A test kit for use in direct agglutination assays which comprises an agglutination reagent according to any one of claims 1 to 5, and a secondary antibody which binds to a new epitope formed by the binding of the analyte binding molecule to the analyte, wherein the secondary antibody is conjugated to an erythrocyte binding molecule.
100. A test kit for use in indirect agglutination assays which comprises an agglutination reagent according to any one of claims 49 to 55 and a soluble analyte binding reagent.
101. A test kit for use in indirect aggulutination assays which comprises an agglutination reagent according to any one of claims 49 to 55 and a soluble analyte binding reagent, further comprising a reference sample containing a known quantity of analyte.
102. The reagent according to claim 49 or 50, wherein said erythrocyte binding molecule is an anti-erythrocyte antibody, or F(ab)2 or FAB' fragment thereof or melittin or a specific binding fragment thereof, and wherein the antibody or F(ab)2 or Fab' fragment thereof is raised against integral membrane protein 1, membrane attached glycoprotein C4, integral membrane glycoprotein, ankyrin, spectrin, glycophorin, glycolipid, glycosphingolipid, protein 4-1 or F-actin.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPI4400 | 1987-09-07 | ||
| AUPI440087 | 1987-09-17 | ||
| US11131387A | 1987-10-22 | 1987-10-22 | |
| AUPI5018 | 1987-10-22 | ||
| US111,313 | 1987-10-22 | ||
| AUPI501887 | 1987-10-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1308652C true CA1308652C (en) | 1992-10-13 |
Family
ID=27157386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000577502A Expired - Lifetime CA1308652C (en) | 1987-09-17 | 1988-09-15 | Agglutination assay |
Country Status (3)
| Country | Link |
|---|---|
| KR (1) | KR920010224B1 (en) |
| AP (1) | AP81A (en) |
| CA (1) | CA1308652C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109804234A (en) * | 2016-08-18 | 2019-05-24 | 皮瑞克生物医学有限公司 | Blood unit test kit |
| CN110672862A (en) * | 2019-09-29 | 2020-01-10 | 迈克生物股份有限公司 | Blood type detection card and preparation method thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4433059A (en) * | 1981-09-08 | 1984-02-21 | Ortho Diagnostic Systems Inc. | Double antibody conjugate |
| JPS59192961A (en) * | 1983-04-15 | 1984-11-01 | Green Cross Corp:The | Reagent for measuring xiii-th factor of blood coagulation |
| JPS62182662A (en) * | 1986-02-06 | 1987-08-11 | Fujirebio Inc | Reagent for detecting virus antibody |
-
1988
- 1988-09-15 CA CA000577502A patent/CA1308652C/en not_active Expired - Lifetime
- 1988-09-16 AP APAP/P/1988/000101A patent/AP81A/en active
- 1988-09-16 KR KR1019880012011A patent/KR920010224B1/en not_active IP Right Cessation
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109804234A (en) * | 2016-08-18 | 2019-05-24 | 皮瑞克生物医学有限公司 | Blood unit test kit |
| CN109804234B (en) * | 2016-08-18 | 2023-03-17 | 皮瑞克生物医学有限公司 | Blood unit test kit |
| CN110672862A (en) * | 2019-09-29 | 2020-01-10 | 迈克生物股份有限公司 | Blood type detection card and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| KR920010224B1 (en) | 1992-11-21 |
| AP8800101A0 (en) | 1990-04-28 |
| AP81A (en) | 1990-04-28 |
| KR890005520A (en) | 1989-05-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5086002A (en) | Erythrocyte agglutination assay | |
| US4894347A (en) | Erythrocyte agglutination assay | |
| Wisdom | Enzyme-immunoassay. | |
| AU583473B2 (en) | Method and products for detection of human t cell leukemia virus | |
| US4292403A (en) | Detection and/or determination of IgM, IgA, IgD and IgE immunoglobulins | |
| JP2636331B2 (en) | One-step assay for antigen-specific antibodies and suitable reagents | |
| US4929543A (en) | Process for the determination of an antibody in human body fluids | |
| US4743678A (en) | Method and products for detection of human T cell leukemia virus | |
| JP2656776B2 (en) | A novel immunochemical assay method for stable glycosylated hemoglobin | |
| US5583003A (en) | Agglutination assay | |
| EP0308242B1 (en) | Agglutination assay | |
| CA1308652C (en) | Agglutination assay | |
| AP156A (en) | Agglutination assay. | |
| US6210906B1 (en) | Specific antibodies to kringle 5 of apo(a) and methods of use therefor | |
| JP3998245B2 (en) | Method and kit for measuring oxidized apolipoprotein AI and oxidized lipoprotein containing the same | |
| AU624580B2 (en) | Reagent, method and kit for agglutination assay | |
| IE61639B1 (en) | Agglutination assay | |
| AU633633B2 (en) | Agglutination assay | |
| US5045448A (en) | Method and products for detection of human T cell leukemia virus | |
| CA2014067A1 (en) | A method for the determination of antibodies against organisms causing infectious diseases in body fluids, agents for this purpose and the use thereof in this method | |
| ISHIZAKA | BIOLOGIC FUNCTION OF IMMUNOGLOBULIN E a TERUKO ISHIZAKA, MD | |
| JPH02264863A (en) | Conjugate of human immunoglobulin fc segment and antibody of mammal other than human being |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |