EP0494210A1 - Analyse par agglutination - Google Patents

Analyse par agglutination

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Publication number
EP0494210A1
EP0494210A1 EP90914469A EP90914469A EP0494210A1 EP 0494210 A1 EP0494210 A1 EP 0494210A1 EP 90914469 A EP90914469 A EP 90914469A EP 90914469 A EP90914469 A EP 90914469A EP 0494210 A1 EP0494210 A1 EP 0494210A1
Authority
EP
European Patent Office
Prior art keywords
analyte
agglutination
reagent
binding
sample
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.)
Withdrawn
Application number
EP90914469A
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German (de)
English (en)
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EP0494210A4 (fr
Inventor
Carmel Judith Hillyard
Dennis Brian Rylatt
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Agen Ltd
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Agen Ltd
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Publication date
Application filed by Agen Ltd filed Critical Agen Ltd
Publication of EP0494210A4 publication Critical patent/EP0494210A4/fr
Publication of EP0494210A1 publication Critical patent/EP0494210A1/fr
Withdrawn legal-status Critical Current

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms

Definitions

  • the present invention relates to a reagent and a method for detecting an antigen, antibody or other analyte in a sample, such as human or animal blood, by an agglutination assay.
  • the invention is also directed to a kit containing the reagents and to processes for preparing the reagents .
  • Immunoassays have revolutionized human diagnostic and veterinary medicine since the introduction of techniques such as the radioimmunoassay (RIA) , first reported by Yalo and Berson (Nature 184:1648 (1959) ) , and the enzyme immunoassay (EIA) which was first reported by Engvall and Perlman (Immunochem. 8: 871 (1971)) and Van Weeman and Schuurs (FEBS Lett. 15:232 (1971)) .
  • RIA radioimmunoassay
  • EIA enzyme immunoassay
  • Immunoassays have enabled extremelyly sensitive measurement of analytes circulating in the blood of a subject, and have allowed the determination of levels of hormones, drugs and other compounds present at very low concentrations (such as picomoles/liter) .
  • Such assays usually involve complex detection systems.
  • the reagents used are generally antigens labeled with an enzyme or a radioisotope, 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 involve several washing steps.
  • 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.
  • red cells are coated with antigen, and contacted with both a soluble antibody and with sample. Sample antigen competitively inhibits the binding of sensitized red cells by the antibody, and hence the agglutination.
  • Agglutination assays may use other agglutinable particles, For example, latex agglutination assays are described in Castelan et al. (J. Clin. Pathol. 21:638 (1968)) and Singer et al. (A er. J. Med. [1956 (Dec)]: 888) .
  • ABMl/ EBM + ABM2/EBM a reagent comprising two or more distinct conjugates, i.e., ABMl/ EBM + ABM2/EBM, where EBM denotes an erythrocyte binding molecule and ABMl and ABM2 are analyte binding molecules specific for different, non-overlapping, non-repeating epitopes of the analyte.
  • An agglutination assay of this type is depicted in Figure 1 (b) .
  • Wardlaw, U.S. 4,695,553 and Guesdon, U.S. 4,668,637 relate to use of "universal" anti-erythrocyte antibodies (and cf. McLaughlin, U.S. 4,683,196), while type-specific antibodies are taught by Lloyd, U.S. 4,678,747, Graham Jr., U.S. 4,358,436, Lu, U.S. 4,550,017, Steplewski, U.S. 4,607,009 and Lennox, W083/03477. Bigtee, Molec.
  • a blood sample may be divided into a plurality of aliquots which are then assayed separately for the presence of different analytes.
  • Mochida discloses the use of monovalent antibodies, or antigen-binding fragments thereof, in certain immunoassays. Forrest (US 4,659,678) mentions that monovalent antibodies cannot form dimers or more extensive com plexes with the antigen; such monovalent antibody-antigen aggregates were said to be capable of interfering with the binding of the antigen-antibody complex to a solid phase support.
  • hemagglutination assays are generally faster, but less sensitive than radioimmunoassays (RIA) or enzyme immunoassays (EIA) .
  • RIA radioimmunoassays
  • EIA enzyme immunoassays
  • the speed and sensitivity of hemagglutinatio assays can be increased by the use of the autologous red cell agglutination technique described in, e.g., U.S. 4,894,347.
  • the advantage of such a technique is that blood does not have to be separated; a single finger prick is sufficient to provide an assayable sample. For analytes which ar large molecules with repeating epitopes, the sensitivity of such an assay is sufficient.
  • the present inventors recognized a need in the art for improved sensitivity in autologous agglutination assays, in particular for detection of hormones such as human chorionic gonadotrophin (HCG) in the early stages of pregnancy.
  • HCG human chorionic gonadotrophin
  • Such assays with increased sensitivity could also serve as alterna tives to inhibition assays in the immunodetection of small molecules.
  • Inhibition assays are inherently less sensitive than direct assays, and the end point is more difficult to define.
  • the invention is useful in assaying low concentrations of antigens lacking repeating determinants .
  • we describe herein a direct agglutination assay wherein an analyte which has two nonrepeating epitopes may be detected without loss of sensitivity due to the formation of multiple "bridges" between an analyte molecule and a single agglutinable particle.
  • a direct agglutination assay for the detection of an analyte in a sample, wherein the analyte includes a first nonrepeating epitope and a second nonrepeating epitope, the assay comprising:
  • a direct agglutination assay for the detection of an analyte in a sample, wherein the analyte includes a first bindin site, the assay comprising:
  • a two site direct agglutination assay for the detection of the simultaneous presence of two different analytes in a sample wherein the first and second analytes each comprise at least one analyte-binding site, the assay comprising:
  • a direct agglutination assay for the detection of an analyte in a sample, wherein the analyte includes a first nonrepeating epitope and a second nonrepeating epitope, the assay comprising:
  • a primary agglutination agent comprising a second primary agglutination reagent:agglutinable particle complex wherein said second primary agglutination reagent is capable of binding to a nonrepeating epitope of said analyte and said first and second reagents binding to different, non-overlapping epitopes of said analyte;
  • a direct agglutination assay for the detection of an analyte in a sample, wherein the analyte includes a first binding site, the assay comprising: (a) providing a first primary agglutination reagent which is capable of binding to an agglutinable particle and to said analyte epitope, and which when bound to said analyte epitope forms a second epitope;
  • a two site direct agglutination assay for the detection of the simultaneous presence of two different analytes in a sample wherein the first and second analytes each comprise at least one analyte-binding site, the assay comprising:
  • a two site direct agglutination assay for the detection of the simultaneous presence of two different analytes in a sample wherein the first and second analytes each comprise at least one analyte-binding site, the assay comprising:
  • a two site direct agglutination assay for the detection of the simultaneous presence of three different analytes in a sample wherein each of said analytes comprise at least one analyte binding site, the assay comprising:
  • a two site direct agglutination assay for the detection of the simultaneous presence of a plurality of different analytes in a sample wherein each of said analytes comprise at least one analyte binding site, the assay comprising:
  • an agglutination assay for the detection of an analyte in a particulate-containing sample wherein the analyte comprises at least three binding sites, the assay comprising: mixing a first conjugate comprising a particulate- binding molecule and an analyte binding molecule capable of binding to first and second binding sites on the analyte with a first particulate-containing sample to form a first mixed sample, mixing a second conjugate comprising a particulate- binding molecule and an analyte binding molecule capable of binding to a third binding site on the analyte with a second particulate-containing sample to form a second mixed sample, mixing the first mixed sample with the second mixed sample wherein agglutination of the combined mixed samples indicates presence of analyte in the first particulate- containing sample.
  • mixing of the first conjugate with the first sample and the second conjugate with the second sample are typically done over 1 - 2 minutes.
  • the first mixed sample and the second mixed sample are typically mixed together for 1 - 2 minutes.
  • a strong positive will agglutinate in 10 - 20 seconds.
  • the sample is divided into two portions
  • a first primary agglutination reagent (Rl) which can simultaneously bind both an agglutinable indicator particle and first, nonrepeating epitope analyte of interest is combined with the analyte in the first portion and with indicator particle (which may be endogenous or exogenous to the sample) .
  • a second primary agglutination reagent (R2) similar in function except that it binds a second, nonrepeating epitope of the analyte, is combined with the second portion and with indicator particle. Th stoichiometry of the reaction is such that there is essentially no uncomplexed analyte in either portion. Also, essentially all the excess reagent is particle bound (P-Rl or P-R2) .
  • the two sample portions are now recombined.
  • the Particle- Reagentl-Analyte complex which has a free second epitope,-reacts with the Reagent2-Particle (P-R2) complex.
  • the Particle-Reagent2 Analyte complex which has a free first epitope, reacts with the Reagentl-Particle (P-Rl) complex.
  • Each reagent molecule is already bound to one particle (from one portion) , but is free to bind only to an analyte molecule complexed to another particle (from the other portion) . Multiple bridges between a single particle and a single analyte molecule are avoided.
  • the assay is adapted to the situation where the analyte initially has only a single, nonrepeating epitope. It relies on the fact that the immune complex of the first reagent and the analyte has epitopes which are not found on either the reagent or the analyte alone.
  • the second reagent binds to such a formed epitope, rather than to an epitope native to the analyte.
  • a third, bispecific secondary agglutination reagent which conjugates a first complex comprising analyte (from the first sample portion) bound to a particle through the first reagent, and a second complex comprising analyte (from the second sample portion) bound to a particle through the second reagent.
  • Each valency of the third reagent may be for either a native epitope of the analyte or one formed by the analyte-Rl or analyte-R2 complex.
  • the specificities of the third reagent are chosen so that it is incapable of conjugating first complex-to-first complex or second complex-to-second complex, it will not be able to conjugate two analyte molecules conjugated already to the same particle, and therefore, will more efficiently contribute to the formation of the desired agglutinate.
  • test kit for use in a direct agglutination assay, for the detection of an analyte in a particulate- containing sample, which comprises:
  • test kit for use in a direct agglutination assay for the detection of an analyte in a particulate- containing sample, comprising a single binding site which comprises:
  • test kit for use in a two site direct agglutination assay for the detection of an analyte in a particulate-containing sample, wherein the analyte comprises at least two binding sites, which comprises:
  • a crosslinking reagent which comprises a dimeric analyte binding molecule capable of binding to a third binding site on the analyte.
  • test kit for use in a two site direct agglutination assay for the detection of an analyte in a particulate-containing sample, wherein the analyte comprises at least two binding sites, which comprises:
  • a crosslinking reagent which comprises a divalent hybrid analyte binding molecule capable of binding to third and fourth binding sites on the analyte.
  • test kit for use in a two site direct agglutination assay for the detection of an analyte in a particulate-containing sample, wherein the analyte comprises at least two binding sites, which comprises:
  • a crosslinking reagent which comprises a divalent hybrid analyte binding molecule capable of binding to a third binding site on the analyte and to a site generated by the binding of the first analyte binding molecule or the second analyte binding molecule to the analyte.
  • test kit for use in a two site direct agglutination assay for the detection of an analyte in a particulate-containing sample, wherein the analyte comprises at least two binding sites, which comprises:
  • a crosslinking reagent which comprises a divalent hybrid analyte binding molecule which is capable of binding to a site generated by the binding of the first analyte binding molecule and to a site generated by the binding of the second analyte binding molecule to the analyte.
  • test kit for use in a two site direct agglutination assay for the detection of an analyte in a particulate-containing sample wherein the analyte comprises at least two binding sites, which comprises:
  • a second conjugate comprising a particulate-binding molecule and a second analyte binding molecule capable of binding to a second binding site on the analyte
  • a crosslinking reagent which comprises a divalent hybrid analyte binding molecule capable of binding to the first binding site on the analyte and to the second binding site on the analyte.
  • a test kit for use in a two site direct agglutination assay for the detection of an analyte in a particulate-containing sample wherein the analyte comprises at least two binding sites which comprises:
  • a crosslinking reagent comprising a divalent hybrid analyte binding molecule capable of binding to the second binding site on the analyte and to a site generated by the binding of the second analyte binding molecule to the second binding site.
  • test kit for use in a two site direct agglutination assay for the simultaneous detection of two different analytes in a particulate-containing sample, wherein the first and second analytes each comprise at least one analyte binding site, which comprises:
  • a crosslinking reagent which comprises a dimeric analyte binding molecule capable of binding to a third binding site on the first analyte and to a fourth binding site on the second analyte.
  • test kit for use in a two site direct agglutination assay for the simultaneous detection of two different analytes in a particulate-containing sample, wherein the first and second analytes each comprise at least one analyte binding site, which comprises:
  • a crosslinking reagent which comprises a divalent hybrid analyte binding molecule capable of binding to a third binding site on the first analyte and to a fourth binding site on the second analyte generated by the binding of the second analyte binding molecule to the second analyte.
  • a crosslinking reagent which comprises a divalent hybrid analyte binding molecule capable of binding to a third binding site on the first analyte generated by the binding of the first analyte binding molecule to the first analyte and to a fourth binding site on the second analyte generated by the binding of the second analyte binding molecule to the second analyte.
  • a crosslinking reagent which comprises a divalent analyte binding molecule capable of binding to a third binding site present on each of the first and second analytes.
  • a test kit for an agglutination assay for the detection of an analyte in a particulate-containing sample wherein the analyte comprises at least three binding sites comprising:
  • test kit for use in a direct agglutination assay for the detection of an analyte in a first particulate- containing sample, wherein the analyte comprises a first binding site and a second binding site, which comprises:
  • a first conjugate comprising a first particulate binding molecule and an analyte binding molecule capable of binding to the first binding site
  • a reagent prepared by mixing/reacting a second particulate-containing sample with a second conjugate comprising an analyte binding molecule capable of binding to the second binding site and a second particulate binding molecule.
  • a test kit for a direct agglutination assay for the detection of an analyte in a first particulate-containing sample, wherein the analyte comprises a binding site which comprises:
  • a reagent prepared by mixing/reacting a second particulate-containing sample with a second conjugate comprising an analyte binding molecule capable of binding to the second binding site and a second particulate binding molecule.
  • a test kit for a two site direct agglutination assay for the detection of an analyte in a first particulate-containing sample, wherein the analyte comprises at least two binding sites which comprises:
  • a reagent prepared by mixing/reacting a second particulate-containing sample with a second conjugate comprising an analyte binding molecule capable of binding to the second binding site and a second particulate binding molecule;
  • cross linking reagent comprises a dimeric analyte binding molecule capable of binding to a third analyte binding site on the analyte.
  • the crosslinking reagent comprises a divalent hybrid analyte binding molecule capable of binding to a third analyte binding site on the analyte and on a fourth analyte binding site on the analyte.
  • the crosslinking reagent comprises a divalent hybrid divalent analyte binding molecule capable of binding to a third analyte binding site on the analyte and to a site generated by the binding of the first analyte binding molecule to the first binding site or the second analyte binding molecule to the second binding site.
  • the crosslinking reagent comprises a divalent hybrid analyte binding molecule capable of binding to a site generated by the binding of the analyte binding molecule to the first binding site, and to a site generated by the binding of the second analyte binding molecule to the second binding site.
  • the crosslinking reagent comprises a divalent hybrid analyte binding molecule capable of binding to the first binding site in the analyte and to the second binding site on the analyte.
  • the crosslinking reagent comprises a divalent hybrid analyte binding molecule capable of binding to the second binding site on the analyte and to a site generated by the binding of the second analyte binding molecule to the second binding site.
  • a test kit for a two site direct agglutination assay for the simultaneous detection of two different analytes in a first particulate-containing sample wherein the first and second analytes each comprise at least one analyte binding site which comprises:
  • a reagent prepared by mixing/reacting a second particulate-containing sample with a second conjugate comprising an analyte binding molecule capable of binding to the second binding site and a second particulate binding molecule;
  • the crosslinking reagent comprises a divalent hybrid analyte binding molecule capable of binding to a third analyte binding site on the first analyte and a fourth analyte binding site on the second analyte.
  • the crosslinking reagent comprises a divalent hybrid analyte binding molecule capable of binding to a third analyte binding site on the first analyte and to a fourth analyte binding site generated by the binding of the second analyte binding molecule to the second analyte.
  • the crosslinking reagent comprises a divalent hybrid analyte binding molecule capable of binding to a third analyte binding site generated by the binding of the first analyte binding molecule to the first analyte, and to a fourth analyte binding site generated by the binding of the second analyte binding molecule to the second analyte.
  • the crosslinking reagent comprises a divalent analyte binding molecule capable of binding to a third analyte binding site present on each of the first and second analytes.
  • test kit for an agglutination assay for the detection of an analyte in a first particulate-containing sample wherein the analyte comprises at least three binding sites which comprises:
  • a reagent prepared by mixing/reacting a second particulate-containing sample with a second conjugate comprising an analyte binding molecule capable of binding to a third binding site on the analyte and a particulate-binding molecule.
  • the particles in the second particulate-containing sample are different from the particles in the first particulate- containing sample.
  • the particulate-containing samples can be whole blood, semen, culture of hybridoma cells, samples obtained from microbial fermentation and tissue culture, for example.
  • the particulate-containing samples can contain synthetic or artificial particles such as carbohydrates, latex (natural or synthetic rubber or plastic) , glass beads, carbohydrates (cellulose) liposomes and metal oxide particles, for example.
  • Figure 1(a) shows the three components of an agglutination assay described in the Hillyard patent: a particle with multiple epitopes, an analyte with two different nonrepeating epitopes, and two primary agglutination reagents, both of which bind the particle, but which also bind the analyte at the different epitopes.
  • Figure 1(b) shows an agglutinate formed by the sequential or simultaneous combination of the aforementioned reaction components.
  • Figure 1(c) reveals how sensitivity can be reduced through the formation of multiple bridges between one molecule of analyte and one erythrocyte.
  • Figure 2 illustrates the present invention.
  • the sample is divided into two portions, each of which is reacted with particles and with one or the other of the two primary agglutination reagents.
  • Figure 3 depicts a variant on the above in which the analyte need only have a single, nonrepeating epitope.
  • the binding of the first reagent to the analyte creates a new epitope which is recognized by the second reagent shown on the right side when the sample portions are recombined
  • Figure 4 illustrates uses of a secondary agglutination reagent. While drawn so as to suggest that the epitope recognized by the reagent is one generated by formation of the analyte- primary reagent complex, this is not in fact required, as may be seen in, e.g., the next Figure.
  • Figure 5 depicts various ways in which the secondary agglutination reagent can bridge two analytes, including (a) binding the same epitopes recognized by the primary reagents, (b) binding a third epitope on each analyte, (c) binding a third and fourth epitope, on each analyte, (d) bridging a third epitope and a complex-formed epitope, (e) binding two complex-formed epitopes, and (f) binding an epitope recognized by a primary reagent and an epitope formed by the complexing of the analyte with the other primary reagent.
  • Figure 6 shows an adaptation of the assay for simultaneous measurement of two different analytes .
  • Figure 7 illustrates use of a first primary agglutination reagent as previously described, in conjunction with a particle- bound analyte binding molecule, the latter replacing the second primary agglutination reagent.
  • Figure 8 depicts an assay for antibody employing a first PBM-ABM reagent wherein the analyte binding molecule is an antigen specifically bound by the analyte antibody and a second reagent which is a carrier particle bound anti-immunoglobulin.
  • this anti immunoglobulin may be covalently bound to the carrier particle (as shown) or conjugated through a particle binding moiety (such as an anti-glycophorin antibody " moiety, in the case of an erythrocyte) .
  • FIGS 9 and 10 are schematic representations of a further type of assay which detects specific immunoglobulin subtypes, such as IgM or allergen-specific IgE.
  • This assay also allows the detection of IgG antibodies to large antigens, which would be difficult to couple directly to the anti erythrocyte antibody.
  • the EBMs can be the same or bind to different sites on the erythrocyte.
  • Figure 9 represents the assay for allergen-specific IgE.
  • the conjugate consists of a bi-specific anti-erythrocyte-anti Ig antibody which binds to only one site on the IgE.
  • the allergen i added as a multivalent moiety, either alone or bound to a carrie such as latex or a protein. Agglutination occurs when IgE antibodies, specific to the allergen are present in the sample.
  • Figure 10 represents the assay for specific IgM.
  • the conjugate consists of a bi-specific anti erythrocyte-anti IgM antibody which binds to only one site on the IgM.
  • the antigen is added as a multivalent moiety, either alone or bound to a carrie such as latex or a protein. Agglutination occurs when IgM antibodies, specific to the antigen are present in the sample.
  • BEST MODE AND OTHER MODES FOR CARRYING OUT THE INVENTION The term "epitope" is used herein to refer to a binding site on a target molecule which is specifically recognized by a binding molecule, and is not limited to binding sites recognized by antibodies.
  • Agglutinable Indicator Particle is used herein to refer to a binding site on a target molecule which is specifically recognized by a binding molecule, and is not limited to binding sites recognized by antibodies.
  • any particle which is capable of agglutination to form detectable agglutinate, and which can be provided in a form for which a specific particle binding molecule exists, may be used in the present agglutination assay.
  • the particle may be a biological or non-biological particle, and it may be natural or artificial in origin. If natural it may be modified to render it more suitable for assay use. If artificial it may be organic or inorganic in composition.
  • a mixture of different sizes or types of particles may be used, and different particles may be used as indicators in different portions of sample.
  • the particles may be colored or fluorescent to facilitat their visualization.
  • the particles may be native to the sample o added to it. If added, they may be added before, during or after the aliquoting of the sample and before, during or after the addition of the agglutination reagent.
  • erythrocytes are preferred indicator particles.
  • the indicator particles be the erythrocytes endogenous to the sample.
  • PBM Particle Binding Moiety or Molecule
  • the particle is an erythrocyte
  • the preferred particle binding moiety or molecule i one which binds erythrocytes.
  • Erythrocyte membranes are lipid bilayers with a variety of proteins. Some proteins occur only on outer portion of the membrane. Other proteins contain a hydrophobic portion allowing the protein to anchor in or pass through the membrane.
  • a membrane-traversing protein also called a transmembrane protein, may also have an intracellular or cytoplasmic portion. Glycophorin A is an example of a transmembrane protein.
  • Known proteins of erythrocyte membranes include Glycophorin A (MN, Ena, Wrb) , Glycophorin B (Ss, 'N', U) , and the minor constituents suc as integral membrane protein 1 (Rh) , membrane attached glycoprotein C4 (Chido & Rodgers) , integral membrane glycoprotei (anion channel), ankyrin, spectrin, protein 4.1 and F-actin.
  • Glycophorin A MN, Ena, Wrb
  • Glycophorin B Ss, 'N', U
  • the minor constituents suc as integral membrane protein 1 (Rh) , membrane attached glycoprotein C4 (Chido & Rodgers) , integral membrane glycoprotei (anion channel), ankyrin, spectrin, protein 4.1 and F-actin.
  • Glycophorin A MN, Ena, Wrb
  • Ss, 'N', U Glycophorin B
  • Erythrocyte binding molecules include antibodies, lectins, and other binding proteins.
  • Erythrocyte membranes contain various antigenic surface constituents including proteins, glycoproteins, glycolipids and lipoproteins.
  • Antibodies which recognize these constituents may be prepared 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 antigen-binding fragments thereof, may be used as an erythrocyte binding molecule (EBM) .
  • the antibody or antibody fragment may be polyvalent, divalent or univalent.
  • a preferred EBM is an antibody (or specific binding fragment thereof) recognizing glycophorin.
  • This molecule comprises 131 amino acids with 16 oligosac ⁇ haride chains.
  • the main fraction (approximately 75% of total) is glycophorin.
  • this is an abundant moiety, which could allow antibody attachment without agglutinating the red cells. It is also readily available in a relatively pure form commercially, (for example, from Sigma Chemical Company) . (See: H. Furthmayr et al., Biophys. Biochem. Res. Comm. 65:113-122 (1975)).
  • conjugates of an erythrocyte binding antibody and an analyte-binding antibody as an agglutination reagent has been constrained by the need to avoid "auto- agglutination.”
  • auto- agglutination is meant the phenomenon attributable to the ability of such a reagent, acting alone, to bind two or more erythrocytes simultaneously, and thereby to cross-link the erythrocytes into an agglutinate.
  • bivalent or pentavalent antibodies notably certain anti-glycophorin mAbs, do not cause significant "auto-agglutination.” It is believed that the anti- glycophorin antibody is non-autoagglutinating for steric reasons; either the antigen binding sites of the intact antibody are able only to bind adjacent epitopes on the same erythrocyte (and not span the distance between two erythrocytes) or only one of the two antigen binding sites can bind to glycophorin at one time.
  • the EBM When the EBM is multivalent, as is the case of a typical antibody, it is desirable that the molecule recognize an erythrocyte membrane constituent which is abundant and well distributed; the binding site should be in such a position that crosslinking between cells is inhibited by steric hindrance, thereby avoiding premature red cell agglutination.
  • crosslinking may be inhibited by the selection of an EBM that recognizes a surface constituent present in sufficient quantity so that the epitopes are sufficiently close for the binding sites on the EBM to be bound by only the one erythrocyte.
  • One aspect of the present invention is thus the use of an intact non-auto-agglutinating anti-erythrocyte antibody (or multivalent binding fragments thereof) in conjugates in erythrocyte agglutination immunoassay.
  • the use of such antibodies is advantageous because the conjugates are easier to prepare in high yields and there is no requirement to make Fab/Fab' fragments, which are difficult to purify to the necessary degree purity from a mixture which also contains agglutinating antibodies.
  • any contaminating F(ab)2 will cause some agglutination. This will reduce the sensitivity of the assay. The problem is avoided, however, by deriving the Fab' fragments only from a non-auto- agglutinating antibody, as taught herein.
  • glycoproteins, glycolipids and other carbohydrate structures on the surface of erythrocytes are recognized by proteins known as lectins, which have an affinity for particular saccharides.
  • Lectins may therefore also be used as EBMs, as contemplated by the present invention.
  • 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, mellitin, and other highly basic peptides.
  • the invention provides a conjugate between mellitin 7-26, or another peptide with similar nonlytic, univalent erythrocyte-binding property with an analyte-binding molecule to form a direct agglutination assay reagent.
  • mellitin 7- 26 can be synthesized much more easily and economically by chemical or recombinant DNA techniques.
  • the present invention is not limited to any particular method of preparing the EBM.
  • the preferred EBMs of the present invention will recognize erythrocyte membrane constituents found on all, or nearly all erythrocytes in a sample, so that erythrocytes endogenous to the blood sample may be used as the agglutinating particles.
  • Such constituents include the so-called "public antigens”.
  • the known blood group specificities are typically conferred by carbohydrate or glycolipid moieties, which are associated with membrane proteins.
  • an EBM 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 ability of a divalent EBM to agglutinate erythrocytes will depend on steric factors such as the mobility of the molecule and the position of the binding sites relative t the lipid bilayer.
  • EBM it is preferable but not necessary that a single EBM be used that recognizes essentially all erythrocytes.
  • EBMs may be used, either in the same or in separate reagents, each of which recognizes a particular group of erythrocytes, but which i aggregate recognize essentially all erythrocytes. While it is preferable that the EBM recognize a natural surface constituent of the erythrocyte, it is possible to coat erythrocytes with a ligand recognized by the EBM, or to treat th erythrocytes so as to expose a normally cryptic ligand. Non-erythrocyte particles may likewise be functionalized so that they may be bound by an antibody or other binding molecule.
  • the sample may be a biological fluid or a non biological fluid.
  • biological fluids obtained in vivo include whole blood, a separated blood fraction, urine, semen, saliva, cerebrospinal fluid, amniotic fluid, ascites fluid, pleural effusion, cyst fluid, pus, tissue extracts, etc.
  • biological fluids obtained in vitro include tissue culture supernatant, such as that of hybridoma cells, or microbial fermentation medium.
  • the sample may also be a non-biological fluid such as drinking water, wastewater, groundwater, or a nonaqueous fluid.
  • the sample is a particulate-containing sample such that the particles are suitable for use as agglutinable indicato particles.
  • the sample may be added to the sample to obtain a "particle-containing sample". This addition may occur before, after or simultaneously with the addition of the primary agglutination reagent as described herein.
  • the analyte may be a substance normally found in blood, such as a blood protein or a hormone, or it may be a foreign substance, such as a drug (including both therapeutic drugs and drugs of abuse) .
  • Typical analytes include hormones (e.g. HCG, LH, FSI1, insulin), enzymes (e.g. amylase, trypsin, CK isoforms) , other polypeptides such as myoglobin, steroids (e.y. sex hormones, corticosteroids, anaboli steroids), drugs (e.g.
  • the analyte-binding moiety or molecule may be any substance having a preferential affinity for the analyte. including monoclonal or polyclonal antibodies lectins, enzymes, or other binding proteins or substances (or binding fragments thereof) .
  • the analyte is an antigen
  • the ABM is usually an antibody, or an antigen-binding fragment of an antibody, such as a F(ab')2, Fab, Fv or VH fragment.
  • the analyte is an antibody
  • the ABM is usually an antigen or hapten recognized by that antibody, or a second antibody raised against the immunoglobulin isotype of the analyte antibody.
  • the assay employs conjugates of a particle binding moiety or molecule (PBM) , especially an erythrocyte binding molecule (EBM) , with an analyte binding moiety or molecule (ABM) .
  • PBM particle binding moiety or molecule
  • EBM erythrocyte binding molecule
  • ABSM an analyte binding moiety or molecule
  • the conjugate may be a single molecule with particle and analyte- binding moieties, or a complex of two or more molecules.
  • the term “molecule” will be used to cover both moieties and molecules, and “conjugate” to cover both a single hybrid molecule with two binding moieties and two conjugated molecules which each have specific binding behavior.
  • the conjugate is obtained by coupling an EBM (or other PBM) to an ABM.
  • the PBM and the ABM may be coupled together directly or indirectly, and by covalent or non-covalent means (or a combination thereof) .
  • covalent or non-covalent means or a combination thereof.
  • the PBM and the ABM may also be coupled noncovalently, for example, by (a) attaching biotin to one and avidin (or streptavidin) 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, or (d) attaching a sugar to one and a corresponding lectin to the other.
  • the binding characteristics should be changed as little as possible. It may be advantageous to provide a spacer moiety between the PBM and the ABM to reduce steric hindrance.
  • the PBM/ABM conjugate may be a hybrid antibody.
  • One method of constructing such a conjugate is the following:
  • Another method for constructing an EBM/ABM conjugate, in the form of a chimeric antibody comprises: (a) treating an anti-erythrocyte mAb-producing hybridoma and an analyte-specific mAb-producing hybridoma with a distinct site-specific irreversible inhibitor of macromolecular biosynthesis; preferably the inhibitor is selected from the group consisting of emetine, actinomycin D, hydroxyurea, ouabain, cycloheximide, edine and sparsomycin;
  • the hybrid or chimeric antibody of the present invention thus comprises two "half molecules," one with specificity for erythrocytes (the EBM) and the other with specificity for the analyte (the ABM) .
  • the antibody' s own disulfide bonds couple the ABM to the EBM to form an appropriate conjugate.
  • Such a hybrid antibody has advantages over a tail-to- tail conjugate as taught in the prior art, which is formed by a bifunctional coupling agent, conjugating an anti-analyte antibody and a univalent fragment of anti-erythrocyte antibody.
  • the advantages include ease of preparation, the preservation of the correct stoichiometry and stereochemistry of both antibodies and the retention of the binding affinity of each fragment.
  • the use of a peptide to bind the erythrocyte has the further advantage that the entire conjugate may be prepared without any need for a bifunctional coupling agent. Instead, a DNA sequence encoding the erythrocyte-binding peptide and the analyte-binding peptide as a single transcriptional unit is provided, and the desired conjugate is expressed as a fusion protein, with the two moieties joined by a simple peptide bond, or with a peptide spacer of desired length.
  • One particularly preferred example is a DNA sequence encoding an erythrocyte-binding antibody fragment (which could be just the binding site, not a complete Fab) and analyte antibody (which could be just the binding site, not a complete Fab) or antigen as a single transcriptional unit is provided, and the desired conjugate is expressed as a fusion protein, with the two moieties joined by a simple peptide bond, or with a peptide spacer of desired length.
  • the divalent peptide may be prepared by direct chemical synthesis.
  • the particles in or added to the samples assayed according to the methods of the present invention have a plurality of binding sites.
  • the first and second primary agglutination reagents may recognize the same or different binding sites (epitopes) on the surface of the particle, and may be prepared by the same or different procedures.
  • the secondary agglutination reagent conjugates the first complex (formed by the particles and analyte of the first sample portion and the first primary agglutination reagent) and the second complex. It thus possesses two complex-binding moieties or molecules (CBM) .
  • CBMs may be the same or different, and they may bind to an epitope found on the analyte originally, or one generated by the binding of an ABM-PBM conjugate to its analyte epitope. This epitope may be the same epitope as is recognized by the PBM-ABM conjugate, or a different analyte epitope.
  • the same techniques may be used to prepare the secondary agglutination reagent as were described for the primary reagent.
  • Test kits for use in the present invention comprise first and second EBM-ABM conjugates as previously described, and may optionally include a third reagent as described.
  • the contemplated direct agglutination assay comprises:
  • the separate mixing according to the above embodiment has been found to increase the sensitivity by as much as 20- to 30-fold as compared to an assay where two conjugates are mixed together with a single blood sample such as in the assay described by the present inventors in Australian Patent Application No. 24182/88 and U.S. Patent 4,894,347.
  • this embodiment relies on the fact that the PBM is present in excess compared with the number of antigenic molecules being detected.
  • the PBM should thus recognize an particle surface epitope which is abundant.
  • the affinity of the interactions does not appear to be a significant factor at analyte concentrations likely to be present in a blood sample.
  • the basis for the increased sensitivity using the two spot system is more likely to be stoichiometric.
  • the molecular weight of HCG is about 50 kDa and that of the conjugate is about 100 kDa. Therefore, many more conjugate molecules are bound to erythrocyte surfaces than there are HCG molecules in the blood sample. Affinity may have some influence; analysis of a series of antibody conjugates with varying affinities for HCG indicated a decrease in sensitivity with decreasing affinity.
  • the anti- erythrocyte antibody has an affinity of 1 x 109 and the highest affinity for HCG was 5.9 x IO 9 .
  • the component When we speak of forming a mixture of several components, the component may be brought together simultaneously, or sequentially in any order.
  • one of the components of a formed mixture is a set of agglutinable particles and another component is a portion of a sample
  • the particles may be endogenous to the sample so that, in effect, sample analyte and agglutinable particles are mixed simultaneously with the remaining reaction component (s) , or the particles may be exogenous, in which case they may be added before or after the division of the sample, and at any stage in the formation of the recited mixture.
  • This assay thus comprises:
  • the second epitope may comprise at least part of the first epitope bound by the first analyte-binding molecule or may result from a conformational change taking place in the analyte molecule upon binding the first analyte-binding molecule.
  • the first and second mixtures may be mixed with secondary agglutination reagent.
  • Use of a secondary reagent makes it less important to employ an agglutinable particle with a large number of non-overlapping epitopes.
  • Six different (and non-limiting) types of secondary reagents which can be used in this version of the assay are described below.
  • a dimeric analyte-binding molecule capable of binding to a third site on each analyte molecule.
  • a divalent hybrid analyte-binding molecule capable of binding to a third site on one analyte molecule and a fourth site on the other analyte molecule.
  • a divalent hybrid analyte-binding molecule capable of binding to a third site on one analyte molecule and to a fourth site which is generated by the binding of an analyte- binding molecule to a binding site on another analyte molecule.
  • a divalent hybrid analyte-binding molecule capable of binding to a site generated by the binding the first analyte- binding molecule (of a primary reagent) to a first analyte molecule, and to a site generated by the binding of the second analyte binding molecule to a second molecule of analyte.
  • a divalent hybrid analyte-binding molecule capable of binding to the first binding site of the analyte and to the second binding site of the analyte.
  • a divalent hybrid analyte binding molecule capable of binding to the second binding site of the analyte and to a site generated by the binding of the second analyte binding molecule to the second binding site.
  • the invention is directed to a two site direct agglutination assay for the detection of the simultaneous presence of two different analytes in a particle containing sample wherein the first and second analytes each comprise at least one analyte-binding site, the assay comprising:
  • the secondary agglutination reagent must be heterobispecific.
  • mixing of the first conjugate with the first sample and the second conjugate with the second sample are performed over a 1-2 minute interval.
  • the first mixed sample and the second mixed sample are typically mixed together for 1-2 minutes.
  • a strong positive test will show agglutination in 10-20 seconds.
  • the blood is mixed with the first ABM-EBM conjugate.
  • a second reagent is added, which comprises an added particle, such as exogenous red blood cells bound to a conjugate of an EBM and a different ABM which binds to a different site on the analyte.
  • the latter reagent can also be an added particle, such as latex or exogenous red blood cells to which an ABM, which binds to a different site on the analyte, has been coupled chemically.
  • particle/ABM and particle:EBM ABM is the same, vis-a-vis the binding of analyte.
  • a whole blood sample containing the analyte (and endogenous erythrocytes serving as the indicator particle) is reacted with an EBM-ABM conjugate, wherein the ABM is typically an antigen specifically bound by the antibody or a second, anti-idiotypic antibody against the analyte antibody.
  • the sensitivity of such an assay may be increased by adding a second, particle-bound, (analyte antibody) -binding reagent.
  • the analyte-binding moiety of this latter reagent is typically an anti-Fc antibody.
  • the particle may be boun to the particle either covalently through its own Fc, or by a particle-binding moiety conjugated covalently or noncovalently to the analyte binding moiety. If all of the analyte in the sample is bound by the first reagent, the second reagent, by binding to the analyte will form additional interparticle connections.
  • the sensitivity of assays for analytes with repeating epitopes is likewise reduced by the formation of multiple bridges between the indicator particle and the analyte, though with such assays only a single PBM-ABM reagent is necessary for agglutination.
  • the method of this invention may be applied to such analytes by deliberately ignoring any repeating epitopes of the analyte, and instead identifying and providing binding molecules for two different nonrepeating epitopes thereof.
  • the present method may provide some advantage in assaying an analyte for which there is an epitope which, while repeating, is uncommon, though the increase in sensitivity will be less pronounced.
  • an assay which detects antibodies of a particular immunoglobulin isotype, such as, for example, IgM, or allergen specific IgE antibodies.
  • This assay also allows the detection of IgG antibodi to large antigens which would be difficult or impossible to coup directly to an anti-erythrocyte antibody.
  • the EBMs can be the sa or bind to different sites on the erythrocyte.
  • One reagent comprises bispecific anti-erythrocyte (or other particle) - anti IgE antibody, which binds to only one site on the IgE molecule.
  • the other is the allergen, a multivalent moiety, added either alone or bound to a carrier substance such as a latex particle or a protein. Agglutination occurs when IgE antibodies specific to the allergen are present in the sample.
  • the reagents are a bispecific anti-erythrocyte - anti IgM conjugate, (which binds to only one site on the IgM molecule) and a multivalent antigenic reagent added either alone or bound to a carrier substance such as a latex particle or a protein. Agglutination occurs when IgM antibodies specific to the antigen are present in the sample.
  • mice were immunized with human erythrocytes and mAbs produced by fusing the spleen cells of immunized animals with mouse myeloma cells, according to methods well-known in the art.
  • Hybridoma supernatants were screened for the presence of the desired antibody by both spin agglutination assay and enzyme immunoassay (EIA) , wherein glycophorin was bound to polystyrene in a microtiter plate.
  • EIA enzyme immunoassay
  • microtiter plates were coated with 10 ug/ml human glycophorin (Sigma Chemical Co., Cat. No. G-73B91) , washed and then incubated with serial dilutions of the mAb. After further washing, the presence of bound antibody was determined by the addition of an enzyme-labelled second antibody (specific for murine immunoglobulin) , followed by the addition of substrate fo the enzyme.
  • the antibody titer was determined to be the greatest dilution of hybridoma supernatant which gave an absorption (A42 reading greater than 0.17 units above background.
  • the hybridoma cells producing mAbs having the desired reactivities were grown in vivo by intraperitoneal injection into mice according to methods well known in the art.
  • the ascites fluids, containing the secreted mAbs, were clarified by centrifugation and screened.
  • RAT 1C3/86 was selected and deposited under the Budapest Treaty at the American Type Culture Collection (12301 Parklawn Drive, Rockville MD, 20852) on 7 September 1988.
  • the cell line has the designation G 26.4.IC3/86, and received the ATCC accession number HB9893.
  • MAbs were purified to homogeneity from ascites fluids by chromatography on hydroxyapatite (Stanker et al., J. Immunol. Meth. 76:157 (1985)) .
  • Monoclonal antibodies reacting with both the beta subunit of hCG and with the complete molecule were prepared by methods well known in the art. Hybridoma supernatants were screened for the presence of desired antibody by enzyme immunoassay wherein the beta subunit of hCG, the alpha subunit and intact hCG were bound to polystyrene in a microtitre plate essentially as described in Example 1.
  • MAb' s were purified to homogeneity from ascitic fluids by chromatography on hydroxylapatite (Stanker ejt al. J. Immunol. Meth 26. 157 1985) .
  • RAT 1C3/86 anti-human red blood cell
  • HCG-2A1/67 and HCG-1A3/63 anti-human hCG
  • 2mg RAT 1C3/86 was digested for 45 minutes with 1% w/w pepsin in a buffer containing 0.1M acetic acid, 70mM sodium chloride pH 3.5.
  • HCG 2A1/67 and HCG 1A3/63 were regenerated by reaction with lOmM mercapthelylamine for 30 minutes at 25 C. Excess reagent was removed by gel filtration chromatography on a TSK-3000 SW column. Separately the thiol form of each of the HCG antibodies and the Ellman's reagent- treated RAT-1C3/86 were incubated for 16 hours at 25 C as described by Brennan et al. The chimeric antibodies thus formed were finally purified by gel filtration chromatography on a TSK-3000 SW column.
  • Reagent 2 10 ug/ml of Conjugate B (a hybrid antibody consisting of "half" erythrocyte-binding antibody, RAT 1C3/86, and "half” HCG specific antibody B) in PBS, pH 7.4, containing 1 mg/ml BSA and 0.01% sodium azide.
  • Conjugate B a hybrid antibody consisting of "half" erythrocyte-binding antibody, RAT 1C3/86, and "half” HCG specific antibody B
  • the method requires two small drops of blood, one of which is first mixed with conjugate 1, the other with conjugate 2. (These two drops are thus the "first and second sample portions" as previously described.) After an incubation period, the two drops are then mixed together.
  • the sensitivity of the test will be lower should the two samples be mixed accidentally before the conjugates have bound t the erythrocytes.
  • the agglutination plate was then rocked gently for 30 seconds and the two pools combined by mixing with a toothpick. The mixture was then rocked gently for a further 2 minutes and the extent of agglutination determined.
  • Reagent 10 ug/ml of conjugate A (as in Example 3), a 0.5% suspension of latex particles (polystyrene, 0.8 um diameter) coated with HCG-specific antibody B, 1 mg/ml BSA in PBS containing 0.01% azide.
  • the method only requires one drop of blood and provides similar sensitivity to the assay of Example 3.
  • 25 ul of test reagent was added and mixed with a small stirring rod, rocked gently for a further 2 minutes, and the presence or absence of agglutination was noted.
  • a peptide from bee venom, mellitin (amino acid sequence: CVLTTGLPALISWIKRKRQQ) , was used as an alternative to the erythrocyte-binding mAb. This peptide binds to the erythrocyte surface without lysing the cell (deGrado W.F. et al., J. A er. Chem. Soc. 103: 679-81 (1981)). The peptide was synthesized by the Merrifield procedure (Hodges et al., Anal. Biochem. 65:241 (1975)).
  • mellitin and a peptide-type ABM may be synthesized as a single unit, having both erythrocyte and analyte binding activity in t single peptide.

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Abstract

L'invention se rapporte à des analyses et à des kits d'analyse servant à détecter la présence de médicaments, d'hormones, de stéroïdes, d'anticorps et d'autres molécules dans un fluide biologique, tel que notamment le sang. Une analyse par agglutination directe est décrite, grâce à laquelle on peut obtenir une meilleure sensibilité lorsqu'on dose des analytes qui manquent d'épitopes répétitifs. Dans cette analyse, l'échantillon est divisé en parties qui sont amenées à réagir séparément avec des réactifs d'agglutination différents mais complémentaires et qui sont ensuite recombinées pour que l'agglutination puisse avoir lieu. Une sensibilité accrue est ainsi obtenue.
EP90914469A 1989-09-25 1990-09-25 Analyse par agglutination Withdrawn EP0494210A1 (fr)

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WO1993024630A1 (fr) * 1992-05-22 1993-12-09 Agen Limited Reactif pour dosages d'agglutination
AU684715B2 (en) * 1992-05-22 1998-01-08 Agen Limited Reagent for agglutination assays
DE4302012C1 (de) * 1993-01-26 1994-07-21 Serosearch Gmbh Entwicklung Un Immunologischer Test
GB2432211B (en) * 2005-11-12 2009-04-22 Platform Diagnostics Ltd Agglutination based assay system
WO2007054714A2 (fr) 2005-11-12 2007-05-18 Platform Diagnostics Limited Analyse par agglutination
AU2013200119B2 (en) * 2005-11-12 2015-06-25 Platform Diagnostics Limited Agglutination assay
EP2660601B1 (fr) * 2007-08-17 2015-10-07 Korea Advanced Institute of Science and Technology Procédés de détection d'interactions moléculaires

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AU543007B2 (en) * 1980-04-15 1985-03-28 Technicon Instruments Corportion Agglutination immunoassay
EP0039195B1 (fr) * 1980-04-28 1986-06-18 Montefiore Hospital and Medical Center Procédé pour la détection d'anticorps
FR2502787A1 (fr) * 1981-03-27 1982-10-01 Human Pharm Sa Laboratoires Nouveau procede de detection de l'hcg et son application notamment dans un nouveau test de grossesse
US4590169A (en) * 1983-11-18 1986-05-20 Beckman Instruments, Inc. Direct particle agglutination immunoassays avoiding false negatives at high antigen concentrations
JPS60257363A (ja) * 1984-06-05 1985-12-19 Dai Ichi Pure Chem Co Ltd Ｆｄｐの測定法
US4900685A (en) * 1987-01-29 1990-02-13 Cytosignet, Inc. Analyte detection in particulate-containing samples
US4894347A (en) * 1987-09-17 1990-01-16 Agen Limited Erythrocyte agglutination assay

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PH30946A (en) 1997-12-23
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IL95789A (en) 1995-12-08
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NZ235450A (en) 1992-07-28
AP9000208A0 (en) 1990-10-31

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