AU624580B2

AU624580B2 - Reagent, method and kit for agglutination assay

Info

Publication number: AU624580B2
Application number: AU24182/88A
Authority: AU
Inventors: Peter G. Bundesen; Carmel J. Hillyard; Bruce F. Kemp; Dennis B. Rylatt
Original assignee: Agen Ltd
Current assignee: Agen Ltd
Priority date: 1987-09-17
Filing date: 1988-10-21
Publication date: 1992-06-18
Anticipated expiration: 2008-09-14
Also published as: AU2418288A

Description

tfcz 1' V ILD J~ S Ref 75988 FORM COMMONWEALTH OF AUSTRALIA PATIJENTS ACT 1952 COMPLETE SPECIFICAT!ON

(ORIGINAL)

FOR OFFICE USE: Class Int Class Complete S-p-cif ication Lodged.' Ac ce pted: Published: 'I Ii* *1 Priority: Related Art: Name and Address of Applicant: Rylatt,Bruce Ernest Address for Service: Ager. Limited 11 Ourbell StreeL Acacia Ridge Queensland 4110 AUSTRAL.IACarmel Judith Hillyard,Dennis Brian Kemp,Peter Gregory Bundesen Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Males, 2000, Australia Complete Specification for the invention entitled: "Reagent, Method and Kit for Agglutination Assay".

rhe following statement ts a Full descrt Ton of this invention, incli~ding the Sbest method of performing it known tc. sl4/us 00, 9 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 ,ndcgenous 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.

0 14 i 0l 1o "Reagent, Method and Kit for Agglutination Assay".

TECHNICAL FIELD The present invention relates to a reagent and a method for detecting an antigen, antibody or other anaiyte in human or animal blood by erythrocyte agglutination. The invention also concerns 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 comoonents from the serum components of the blood by centrifugation and/or clotting, prior to assay, This presents several potential problems. Firstly, such an assay is not suited to testipo being conducted under field conditions. In many veterinary situations a quick test in the field is 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 S diseases in Third World countries present a situation where simplicity and S low cost are of the essence.

Secondly, in certain pathologic conditions, separation of the blood s" amples becomes difficult. Blood taken from patients suffering conditions such as Waldenstrom's macroglobulinemia is difficult to separate into serum and cell fractions making an assay which can be conducted on whole blood highly desirable.

Thirdly, blood samples are often used for testing for the presence of S' highly contagious and potentially dangerous disease states. In these cases it is preferable that as little handling and processing of the samples as possible is undertaken in order to minimize the risk to personnel conducting the assay, Further, certain conditions make the provision of i. 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 184, 1648, and the enzyme immunoassay or EIA which was first reported by Engvall and Perlman (1971) Immunochem 8, 871 and Van Weeman and Schuurs (1971) FEBS Letters 15, 232, pL 4, 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 involve several washing steps. Most immunoassays for the detection of analytes in blood are currently of this nature. Thus, whilst these assays a,'e sensitive, i 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 type described by Gupta, ft 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 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 and consequently requires the use of a centrifuge or filtration system.

Latex agglutination assays are described in eaOVSF€.

E:v A Caste a-rf- ^Fr9+--4 82# ~-aseln et al,, J. Clin. Pathol.

(1968), 21, 638; and Singer Plotz AM. J. Med. [1956 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, US 3,882,225, who says that it is difficult to standardize indicator erythrocytes.

Molinaro, US 4,130,634 describe: an assay for an antigen which employs antibody-coated 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 possibility of using a hybrid antibody with one binding site specific for the antigen and the other specific for the red :/295c blood cell.

Chang, US 4,433,059 discloses an agglutination immunoassay reagent in which two antibodies are covalently linked "tai -to-tail", so as not to alter their specificity. One antibody is specific for an antigen borne by an indicator sub:tance, such as an erythrocyte. This antibody is preferably univalent to avoid nonspecific agglutination. The other antibody is divalent and is specific for the analyte, In preparation for the assay, fresh erythrocytes are coated with the conjugate. The double antibody conjugate-coated RBCs are then incubated with 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 cytotoxic 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 targef: cel 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, Wardlaw, US 4,695,553 teaches use of a monoclonal antibody against a universal erythrocyte antigen as a RBC agglutinating agent to clarify the interface between red blood cells and white blood cells in centrifuged whole blood. He prefers use of antibodies against glycophorin or against H antigen, but also mentions the possibility 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, 20: 1353-1362 (1983)] describes the production and testing of four monoclonal antibodies against glycophorin A. The general concept of using In an immunoassay an antibody which reacts with an antigenic ADC/295c -4determinant 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, Lloyd, US 4,678,747; Graham, Jr., US 4,358,436; Liu, US 4,550,017; Steplewski, US 4,607,009; Lnnox, W083/03477, These antibodies are useful for blood typing Lecause they bind to antigens found only in certain blood cell populations, while for the purpose of this invention, it s desirable to use antibodies (or mixtures thereof) which bind to essent illy all erythrocytes.

Zuk, US 4,594,327 recogizes the desirability of performing an immunoassay directly on whole blood samples. In his method, the sample is contacted with both an insolubilized, an:lyte-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 RBC binding agent are not coupled together, and the assay disclosed is not an agglutination assay, The problem, in an agglutination immunoassay, of nonspecific S agglutination of erythrocytes by antl-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; Duernieyer, US 4,292,403 and Goldenberg, US 4,331,647 are of interest as demonstrating the use of specific binding fragments of antibodies as substitutes for intact antibodies in assays. The construction of heterobifunctonal 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 antibodies or binding fragments thereof in certain immunoassays. Forrest, US 4,659,878 mentions that monovalent antibodies cannot form dimers or more extensive complexes with the antigen; such aggregates were said to be capable of Interfering with the binding of the antigen-antibody complex to a solid phase support.

DESCRIPTION!OF THE.INVENTION According to a first embodiment of this invention there is provided an agglutination reagent for direct detection of an analyte in a blood p~LA"X sample, comprising an erythrocyte binding molecule attached to an analyte Si~ binding molecule, wherlin the erythrocyte binding molecule is attached to A'TE J CLB/0014e j 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.

According to a second embodiment of this invention there is provided an agglutination reagent 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 Sdoes 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 or fragment essentially os not auto-agglutinate erythrocytes, or a fragment of such an antioody According to a third embodiment of this invention there is provided 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.

According to a fourth embodiment of this invention there is provided 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.

According to a fifth embodiment of this invention there is provided a direct agglutination assay for the presence of an analyte in a blood Llic. sample containing erythrocytes which assay comprises mixing the sample Z ith an agglutination reagent according to any of the first to fourth q /0014e -Sbembodiments and observing whether the erythrocytes are agglutinated and correlating the agglutination with the amount of analyte present in the sample.

According to a sixth embodiment of this invention there is provided 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 erythrocyte membrane but incapable of lysing tirythrocytes, 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.

According to a seventh embodiment of this invention there is provided a direct agglutination assay for the presence of an analyte in a whole blood sample from a subject which comprises contacting a blood sample co-taining erythrocytes endogenous to the subject with an agglutination reagent which comprises a conjugate of a multivalent monoclonal antibody which binds to erythrocyte membranes or a binding fragment thereof, and 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 agglutination with the presence or amount of analyte present, According to an eighth embodiment of this invention there is provided an agglutination assay for an analyte lacking repeatinC 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.

According to a ninth embodiment of this invention there is provided an agglutination assay for an analyte lacking repeating epitopes which comprises incubating a sample which may contain such analyte, ZLAV erythrocytes, a conjugate of an erythrocyte binding molecula and an B/0014e

P.-

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.

According to a tenth embodiment of this invention there is provided 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 bindir: )lecule wherein the conjugate is a heterobifunctional antibody or a heterobifunctional binding fragir-nt 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 detectable amount of homobifunctional erythrocyte-binding antibody, but said homobifunctional erythrocyte-binding antibody essentially does not auto-agglutinate a erythrocytes, According to an eleventh embodiment of this invention there is 25 provided an agglutination reagent for indirect detection of an analyte in S 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 a. 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.

According to a twelfth embodiment of this invention there is provided an agglutination reagent which comprises a conjugate comprising an erythrocyte bindrig molecule conjugated with an analyte analogue I wherein said conjugate does not substantially alter the binding characteristics of the erythrocyte binding molecule (EBM) jr the analyte E B/0014e 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.

According to a t.,elfth embodiment of this invention there is provided 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.

According to a thirteenth embodiment of this invention there is provided 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 t, the eleventh or twelfth embodiment 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, According to a fourteenth embodiment of this invention there is provided an Indirect agglutination assay for the presence or amount of the analyte in a sample which comprises forming a mixture of sample, erythrocytes, an agglutination reagent which comprises a conjugate comprlsing 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, permitting said conjugate to compete with sample analyte for the analyte binding sites of the analyte binding reagent, observing whether agglutination occurs, and inversely correlating the degree of agglutination with the amount of analyte present, According to a fifteenth embodiment of this invention there is .^LIAN provided an Indirect agglutination assay for the presence of an analyte I n a whole blood sample from a subject which comprises contacting a blood sample containing erythrocytes endogenous to the subject with (i) an agglutination reagent which comprises 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, observing whether agglutination occurs, and inversely correlating the degree of agglutination with the presence or amount of analyte present.

According to a sixtoenth embodiment of this invention there is provided a test kit for use in direct agglutination assays which comprises 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 a secondary binding molecule which binds to a new epitope formed by the binding of the analyte binding molecule to the analyte.

According to a seventeenth embodiment of this invention there is provided a direct agglutination assay test kit comprising an agglutination reagent according to any one of the first to fourth embodiments and a reference solution containing a known quantity of analyte, According to an eighteenth embodiment of this invention there is provided a test kit for use in indirect agglutination assays which comprises an agglutination reagent according to the eleventh or twelfth embodiment and a soluble analyte binding reagent.

The present inventors recognized that there was a need for a method which can be used in the laboratory and in the field, particularly in Third World Countries where there is a lack of medical testing facilities for analysis of different types of analytes in whole blood, As indicated above, earlier methods require separation of the blood cells from serum or plasma and are therefore difficult and in many cases impossible to 0014e

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o cc o 0 implement in the field.

If erythrocyte-binding molecules are coupled to specific analyte-binding moleciles, then the resulting conjugate could be used to bind both endogenous erythrocytes, and analytes present in a blood sample.

The present invention results from the finding that when such a complex is exposed to a blood sample, agglutination of t, ,throcytes endogenous to that sample will serve as an indicator of the presence of the relevant analyte (usually, an antigen or antibody) due to cross-linking of erythrocytes with the analyte.

Advantages of endogenous RBCs i) Simplifies current assay procedures no need to centrifuge sample; whole blood, collected in the presence of a suitable anticoagulant, is used instead of serum or plasma.

for samples from patients with infectious diseases, such as AIDS or hepatitis, there is minimal sample nandling.

appropriate for mass screening programs as conducted by the World Health Organization in third world countries, whose facilities are limited, the assay is very rooust; there is only a single reagent, which is stable in the presence cf a bActeriostatic agent such as 0.01% sodium azide, S can be used as a field test by veterinary practitioners, when the appropriate animal red cells 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 druqs and patient compliance.

it also has possible use as an "over-the-counter" self testlng assay.

the only equipment needed is a mixing stick, glass or plastic slide, lancet and possibly a microcapillary.

ii) Advantages over exogenous erythrocttes include: no pretreatment of erythrocytes. Patent 4,433,059 uses blood group 0 negative cells, which have been spun down, reacted with antibody conjugate for 15-30 minutes and washed 3x in PBS.

Patent 4,668,647 uses sheep red blood cells, whic' have been

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000b00 0 0 ADC/295C 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 notes that samples have to be heat inactivated to avoid interference due to complement. Rabbit serum and oovine albumin must also be added to minimize other non-specific reactions. None of this is necessary with the present system, 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 tre selection of an erythrocyte binding molecule such that incubation of conjugate with endogenous erythrocytes will not cause agglutination of the erythrocytes unlesl anaiyte 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 bi ,ing 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 repeating °o determinants, using two conjugates, one bearing an analyte-specific binding oO° 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 Drawings Fig. 1 is a schematic representation of erythrocytes showing positive ano negative agglutination results with antibody complexes in the presence and absence of itigen respectively.

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.

ADC/295c -7-

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Fig 3. is a schematic representation depicting erythrocyte agglutination and inhibition of erythrocyte agglutination due to presence of analyte or antigen.

Fig 4. is a schematic repesentation 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 characteristics of the binding portions. The reagent is non-agglutinating when incubated with endogenous erythrocytes in the absence of the analyte.

Erythrocyte Binding Molecules Erythrocyte membranes contain various antigelic 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 °o thereof, as 1 .unogens. These antibodies may oe monoclonal or )olyclonal in nature. Eith- 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 structures on the surface of erythrocytes are recognized by chemicals known 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 S molecules with an affinity for the lipid bilayer of the membrane, Examples

S

1 of such molecules are: protamine, the membrane binding portion of the bee venom, melittin, and other very basic peptides.

S The preferred EBMs of the prssent 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 bilay^rs with a variety of proteins either on the surface or with a hydrophobic portion allowing the pro ein to ADC/295c -8anchor in 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 ai EBM should recognize eith-r the protein part of a membrane glycoprotein constituent, which is common t- all erythrocytes of a particular species or another common structure. The ability of a bivalent EBM 'o agglutinate red cells will depend on steric factors, such a, the mobility of the molecule and the position of the binding site above the lipid bilayer.

The proteins of erythrocyte membranes include:- glycophor'n A (MN, Ena, Wrb), glycophorin B (Ss, U) and the minor constituents, integral membrane protein 1 (Rhesus), membrane attached glycoprotein C4 (Chido Rodgers), integral membrane glycoprotein (anion channel), glycolipids (Lewis), glycosphingolipids (ABH, li, P, Tk), ankyrin, spectrin, protein 4-1, F-actin. [The associated blood group factors are in parentheses.] The following publications are incorporated by reference: S 1. The red cell membrane, S.B. Shohet E. Beutler, In: Hematology, 3rd ed. Eds: Williams, Beutler, Erslev Lichtman, 1983.

(Review of all erythrocyte membrane antigens.

2. The red cell membrane skeleton. V.T. Marchesi, Blood, 61, 1-11, 198,.

(Review of the skeleton proteins) An especially preferred EBM is one recognizing glycophorir,, When erythrocyte slaloglycopeptides are extracted from membranes, the main fraction (approximately 75% of total) Is glycophorin. This molecule comprises 131 amino acids with 16 oligosaccharide chains. Thus, this is an abundant moiety, which could allow antibody attachment without agglutinating the red cells. It i aiso readily available in a relatively pure form commercially, from Sigma Chemical Company. (See "Fractionation of the Major Sialoglycopeptides of the Human Red Blood Cell Membrane" H. Furthmayr, M. Tomita V.T. Marchesi. BBRC 65, 1975, 113-122).

When the erythrocyte binding molecule is muit ~if ft as in the case of a normal antibody, it is desirable that the molecule recognize an erythrocyte membrane constituent which is abundant and well-distributed, and the binding site should be in such a position that crosslinking between cells is Inhibited by steric hindrance, thereby avoiding premature red cell ADC/295c -9agglutination.

i Alternatively, crosslinking may be inhibited by the selection of an EBM that recognises 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 the one RBC.

It is preferable, but not necessary, that a single EBM be used that recognizes essentially all erythrocytes. Several EBMs may be used, either in the same or in separate conjugates, each of which recognizes d particular group of erythrocytes, but which in aggregate recognize essentially all erythrocytes.

While it is preferable that the Eil recognize a natural surface constituent of the erythrocyte, it is po:,ible to coat erythrocytes with a ligand recognized by the EBM, or to treat the erythrccytes so as to expose a normally cryptic ligand.

Analytes This invention is not limited to the detection of any particular analyte. 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, sJch as a drug (includingi both therapeutic drugs and drugs of abuse), or an organism, 00" such as a virus (by recognizing a virus coat protein) bacterium, protozoan, o fungus, or multicellular parasite heartworm).

The analyte may have repeating epitopes, recognizable by one analyte S binding molecule, or unique epitopes, where a mixture of analyte binding molecules is necessary, However, analytes which can only be bound b one ABM at a time, may also be detected.

Analyte Binding Molecule 'he analyte binding 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 fragmeats thereof), Where the analyte is an antigen, the ABM is usually an antibody. Where the analyte is an antibody, the ABM is usually an antigen recognized by that antibody. When the analyte to be detected has no repeating epitopes, two or more ABMs are required with different specificities for the analyte. The reagent in this case will be either a S mixture of EBM bound to ABMI and EBM bound to ABM2, or EBM with both ABMs attached.

The analyte binding molecule need not bind the analyte directly. ip C 5c -thM ma-y b d A C -10fi V_

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-g2wthhorm Coupling of EBM and ABM TheEBMandtheABMmay be coupled together directly or indirectly, and by covalent or non-covalent means (or a combination thereof). Where multiple EBN~s or ABMs are used, EF.1Is or ABMs may be coupled together, with one or more AB~s coupled directly to an EBM. The following table summarizes some of the covalent coupling methcds known in the art.

Heterobi functional I. SPDP (N-Succinimidyl-3,2-(pyridyldithio)prooionate) Neurath, et al., 1981, J. Virol., Meth., 155-165, 2. MBS (m-maleimidobenzoyl-N-hydroxysuccinimide e~ter) Kitagaw, et al., 1976, J. Biochem,, 79, 223-236.

3. SIAB (N-succi nimi dyl-4-iodoacetylaminobenzoate) Weitman, 9t al., 1983., Bio. Techniques, 1, 148-152.

Selective Bifunctional 0 goP-i sothiocyanatobenzoylchloride a US Patent 4 680 338 Bifunctional 1. BSOCOES off Bis[2-(succinimidooxycarbonyloxy)ethyllsulphione Zarling, et al., 1980, J. Immunol., 124, 913-920 2. BS Bis(sulphosuccinimidyl )suberate Staros, 1982, Biochemistry, 21, 3950-3955.

Other 1. Glutaraldehyde 44*0 Avrameas, 1969, Immunochem., 6, 43.

2. Perlodate Oxidation Nakane and Kawoi 1974, 3. Histochem. Cytochem,, 22, 1084-1091 'a 3. Carbodlimide of the foregoing methods of covalent coupling, conjugation with SPOP is preferred.

The EBM and the ABM may also be coupled noncovalently, for example, by attaching biotin to one and avidin (or strepavidin) to the other), 295c -1 attaching an anti-antibody to one, which then binds the other, (c) attaching Protein A to one, which then binds the F portion of the other, and attaching a sugar to one and a correspondiig 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: preparing fragments of a selected antibody by pepsin digestion; reducing and treating the fragments with Ellman's reagent to produce Fab' fragments of the selected antibody; thiolysing a selected specific antibody or a selected erythrocyte antibody; and coupling the thioylated Fab' fragment to the Ellman's reagent treated Fab' fragment to produce a chimeric anti-erythrocyte antibody-antigen specific antibody conjugate.

Another method is ,et forth below: treating af, anti-erythrocyte monoclonal antibody-producing hybridoma and an antigen specific monoclonal antibody-producing hybridoma with a distinct site-specific irreversible inhibitor of macromolecular biosynthesis; fusing the two different monoclonal antibody-producing hybridomas with polyethylene glycol cloning the fused cells either in soft agarose or by limiting dilution; selecting cloned heterohybridomas secreting chimeric anti-erythrocyte antibody-antigen specific antibody with a screening assay appropriate to the antibodies, purifying the antibody product by affinity purification to free it from non-hybrid antibodies.

Preferably the inhibitor is selected from the group consisting of emetine, actinomycin D, hydroxyurea, ouabain, cycloheximide, edine and sparsomycin.

The chimeric antibody may be two half-molecules, one with specificity for erythrocytes (the EBM) and the other with specificity for the analyte (the ABM). In this case the disulfide bonds of the antibody couple the ABM 4;pa 0a 4 II 0 rrc ADC/295c 1 to the EBM to form 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 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 binding 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 molecules of antigen act as a bridge between proximate erythrocytes. For assaying such analytes, it is preferableto employ a reagent comprising two or rrore distinct conjugates, ABMI/EBM ABM2/EBM where ABM1 and ABM2 bind to different, non-overlapping epitopes of the analyte. One might instead use a more complex single conjugate, ABMI/ABM2/EBM, where the spatial conformation is unlikely to favor the binding of both ABMs on the same conjugate molecule to the same analyte moleculp, Erythrocyte Agglutination 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 intibody, 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 coated with antigen, and contacted with both a soluble antibody and with sample. Samole antigen S4 competitively inhibits the binding of the sensitized red cells by the Santibody, 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 S or an indirect agglutination assay format. However, unlike conventional assays, it is not necessary to precoat erythrocytes with antibody or antigen. Rather, the reagent may be added to a blood sample containing endogenous erythrocytes, whereupon it will sensitize the cells, rendering them able to bind sample analyte (a direct assay) or to compete with sample analyte for a soluble analtye-binding molecule ule -lse (an indirect assay).

295c -13- For some snali circulating molecules such as synthetic or natural steriods, digoxin, theophylline, etc., or drugs of abuse, i 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 addition 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 unconjugated ABM. (The two stages may be reversed). If analyte is present in the blood sample, the specific binding of the AdM 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 anayte, 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 S 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 to the small circulating antigen would be coupled to the erythrocyte binoing molecule, The second (secondary) monoclonal antibody would be incubated with the above conjugate and the analyte and would be capable of binding to a new o oa antigenic determinant comprised of an overlapping region of the first S monoclonal antibody and the antigen that exists only when the first S"a mo'oclonal antibody binds antigen, Thus, the second monoclonal antibody 4o4o acts as the erythrocyte "bridge", finally causes cross-liaking between different red cells allowing agglutination to occur, This method, of S course, is not restricted to monoepitopic analytes, Because of spatial conformation, it may be difficult for a single secondary antibody molecule to bind simultaneously to two conjugate: S" analyte complexes. Thus, It may be preferable to conjugate the secondary antibody with an erythrocyte binding molecule.

In stating that a sample is to be incubated with a plurality of reagents it Is to be understood that the contact may be simultaneous or 'A /295c -14sequential, without limitation to any particular order or duration of contact.

EXAMPLE 1 Preparation of erthyrocyte binding molecule (anti-glycophorin antibody) Immunization and Screening Procedure Mice were immunized with human red blood cells and monoclonal a p\e-emr antibodies produced by fusing the4se- 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 performd by a modification of Wyatt Street, Aust. J, Med. Lab. Sci, 4 48-50. 50 p1 of cell culture supernatant was mixed with 50 pl of a 1% red blood cell suspension in a microtitre plate. For this example, antibodies which bound glycophorin, but did not agglutinate, were selected. The reaction of monoclonal antibody and glycophorin was determined by enzyme immunoassay. Microplates 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 largesc dilution of monoclonal, which gave an A420 reading greater than 0.1 00 units above background.

Of 384 wells, 40 primary clones were chosen. These gave either a positive spin agglutination assay, a response to glycophorin on EIA or both, EIA Spin agglutination Number of clones O Negative Positive 4 Positive Positive Positive Negative 16 a a Subsequent absorption studies were performed to confirm that the S antibodies recognized a glycophorin domain exposed on the red cell surface.

The results of the screening assays on ascitic fluid are listed below: cFF ADC.295c5 -K Ascitic Fluid Titre Spin Red Cell Clone Agglutination Glycophorin EIA Absorption Test RAT 1D3/167 512000 <1000 Positive RAT 3D6/5 6400 1024000 Positive RAT 1C3/86 <1000 1024000 Pcsitive RAT 381/172 256000 2000 Positive RAT 303/22 4000 1024000 Positive RAT 3D5/61 128000 1024000 Positive RAT 1A2/187 <1000 256000 Positive RAT 2A2/187 <1000 128000 Positive RAT IA3/129 <1000 12800 Weak RAT 1C4/5 <1000 128000 Positive RAT 4C3/13 <1000 128000 Positive RAT 381/70 <1000 517000 Positie RAT IC3/86 has been deposited under the Budapest Treaty, with the B 9893 designation G 26.4.1C3/86, ATCCA at the American Type Cultu-e Collection, 12301 Parklawn Drive, Rockville MD, 20852, USA on 7 September 1988.

Purification of RAT 1C3/86 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 conjugate The spread of the human Immuno deficiency virus (HIV-1) has become a Si;ajor 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 antl-HIV antibodies.

We have made use of the patient's own red cells to provide a S 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 DC/295c '6- OH~i' in the presence of antibodies to this antigen. The synthetic peptide antigen derived from gp41 of HIV-1 (residues 579-602), was chosen or the basis of the Welling procedure, FEBS LETT. 188: 215 (1985) and corresponds with the region identified as a major epitope recognised by antibodie: from approximately 98% of AIDS patients. [Wang et al Proc. Nat. Acad. Sci. USA 83: 6259 (1986)].

Synthetic peptides were synthesized using the Merrifield procedure Arna\ [RS Hodges and RB MerrifieldAm. Biochem. 65, 241 (1975)] with the aid of an Applied Biosystems Model 430 synthesizer using double couoling cycles supplied by the manufacturer. 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-NO 2 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 anisole [JM Stewart and JD Young, Solid Phase Peptide Synthesis, pp44 and 66, NH Freeman, San Francisco (1966)]. The crude peptide was precipitated with diethylether, washed with ethylacetate, and extracted with 60% acetonitrile in 0.1% trifluoroacetic acid Synthetic peptides were purified by preparative reverse phase chromotography (Amicon C1 resin, 250A pore size 25 x 400 mm), eluting with i gradient of 1,000m 0 to 60% acetonitrile in 0.1% trifluoroacetic acid, The synthetic peptide was approximately 95% pure as judged by analytical reversed phase SHPLC and by quantitative amino acid analysis following acid hydrolysis.

1. SPDP labelling of the erythrocyte binding Ab (RAT 1C3/86) To 0,25 ml of 13.8 mg/ml RAT 1C3/86 was added 12.5 pl of 2 mg/ml SPDP in dimethyl formamide and the reaction was allowed to proceed for 1 S hour at 25°C, Unreacted SPDP was removed by gel filtration on Sephadex G and the level of SPDP labelling (1.4 moles/mole) was determined, 2, Reduction of peptide 3.2 Peptide 3,2 (sequence RILAVERYLKDQQLLGIWGCSGK, corresponding to residues 579-601 of the major coat protein of HIV 1) was dissolved in 1 ml of 100mM Tris HCI mM EDTA pH 8,0 and reacted with 10 pl of 2-mercaptoethanol for 45 minutes at 40*C, The reaction was terminated by the addition of 4 drops of trifluoroacetic acid (TFA) and 1 mi of aqueous 0.1% TFA, The mixture was applied to a Sep-pak (Waters) C 18 cartridge that had been treated with 20 ml of 60% acetonitrile t stei and -17 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 100mM potassium phosphate, 100mM sodium chloride and 4M guanidine HC1 pH 7.4 and mixed with 2.2 mg 34 SPDP of labelled antibody in the same buffer, but without guanidine HC1. The flask was incubated overnight at The degree of substitution of the antibody Influenced the solubility of the conjugate; 20 moles peplide 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 Chromatography Unreacted peptide and SPDP by-products were removed by gel filtration on a Superose 6 column (Pharmacia) in joe-pe buffered saline and antibody containing fractions were pooled and stored at 4 0 C after addition of 0.01% sodium azide as a preservative, S 5. Preparation of reagent for assay o, Two volumes of conjugates were mixed with one volume of a 10 mg/mi solution of an unrelated monoclonal antibody (Bruce 5) prepared as described in Bundesen, et al,, Vet. rmmLiin. Immunopath. 8, 245-260, 1985.

S Assay procedure For assay, 10 p1 (f heparinized whole blood was placed on a glass o, slide. 30 pl of reagent was added and mixed. The slide was rocked for up to th.ee minutes and the presence or absence of agglutinatlon noted, Nine independent peptide/antibody conjugates were prepared and found to be active in agglutinating seropositive patient's red blood cells, Active conjugate was also prepared using m-Maleimidobenzoyl-N-hydroxysuccnimide ester as the cross-linking reagent.

Results were at least comparable in accuracy to those observed with S an enzyme4M'aas 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 orythrocyte assay.

Control blood samples comprised ELISA negative blood samples and C/295c -18i ELISA positive samples from infected patients. HiV positive patients were confirmed western blot positive by the Victorian State Reference Laboratory. Fairfield hospital patients were negative either by western blot or EIA (AbbottLaboratories). Blood donors were tested by EIA (Genetic Systems). False positive or negative values are given in parnntheses and were verified by EIA or western blot analysis.

TABLE 1 Autologous Red Cell Agglutination Test Agglutination EIA Test Test +ve -ve +ve -ve HIV +ve patients 42 43 0 Fairfield Hospital patients 63 0 66 Healthy blood donors 873 872 In order to evaluate the specificity of the test a series of synthetic peptides corresponding to other regions of the HIV-1 envelope proteins was tested for their capacity to inhibit agglutination reaction (Table 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 :ositive 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, ADC/295c 9- 1 TABLE 2 Specificity of peptide inhibition of agglutination Added Synthetic Inhibition of Peptide (sequence) Agglutination none 0% gp41 (579-601) RILAVERYLKDOOLLGIWGCSGK 100% gp4l (572-591 GIKQLARILAVERYLKADOO 0% gpl2O (193-200) ASTTTNYT gp120 (105-117) HEDIISLkJDQSLK 0%/ gpl2O (101-118) VEQMHEDIISLOSLKP 0 gp120 (105-129)Y 1 2 9 HEDIISLWSOSLKPAVKLTPLCVSJY 0% EXAMPLE 3 Preparation of Chimeric Antibodies (anti-L: ty ppoin/ anti-human 0-dimer) and use in assay for D-dime' Monoclonal antibodies RAT 1C3/86 (anti-human red blood cell) and DD-1C3/108 (anti-human D-dimer as described by Rylatt, et 1983) Thrombosis Res 31, 767-778) were digested with pepsin essentially as described by Hackman, et 1981, Immunology, 15, 429-436, and purified by chromotrography on a TSK-3000 SWI column. 2 mg RAT 1C3186 was digested for 45 minutes with 1% w/w pepsin in a buffer containing 0.IM acetic acid, sodium chloride pH 3,5. Meanwhile, 2 mg 00-103/108 was digested with 1% w/w pepsin for Z hours in the same buffer, The reactions were La terminated by the addition of 1,5M Tris to raise the pH18, U.The F (ab) 2 fragments were purified by gel filtration chromatography onITSK-.3000 SW Scolumn.

*to 0 Reduction of the FVab)2, and subsoquent 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 101 mercaptoethylanine, In the presence of 10mM sodium arsenite, for 16 hours at 25 0 C. The Fab fragments were stabilized by reaction with 5.1Choi (2-nitrobenzolc acid) (Ellman's reagent) for 3 hours at 250C. The Fab fragment was then purified by gel filtration chromatography on a TSK-3000 SW column.

The thiol form of DO-1C3/108 was regenerated by reaction with l0mM mercaptoethylamine for 30 minutes at 250C. Excess reagent was removed by gel filtration chromatography on a TSK--3000 SN column. A mixture of the th'ol DD-1C3/108 ar. e Ellman's reagent treated RAT IC3/86 was incubated for 16 hours at 2 s described by Brennan, et al. Finall., the chimeric antibouy was purified b, further gel filtration chromatography on a TSK-3000 SN column.

Preparation oLf reaggent Two volumes of 0.1 mg/mi chimeric antibody was mixed with one volume of 7.5 mg/ml unrelated monoclonal antibody (Bruce Assay procedure For assay, Ir pl of heparinized whole blood was placed on a glass slide. 30 p1 of reagent was added and mixed. The slice was rocked for three minutes and in the presence of D-dimer agglutination was observed.

EXAMPLE 4 Preparation of SPDP-conjugated D0goxin/Anti-gycophorin antibody conjugate Preparation of Dihxin/Ab conjugate 1) Preparation of periodate oxidized digoxin.

2 ml 100mM sodium periodate was added slowly, dropwise, to 40 mg of digoxin (Sigma), suspended in 2 mi 95%4a-tha- and the reaction was allowed to continue for 30 min at 37 0 C. The reaction was stopped by the addition of 60 p1 oflM ethandiol, Finally, the Schiff's base intermediate was stabilized by the addition of 2 ml 40mM cystine min: 37 4 C) and subsequent reaction with 1 ml of 15 mg/ml sodium borohydride (16h: 2) Reduction of cystine/digoxin conjugate.

3 ml of cystine/digoxin conjugate was reduced by the addition of pi mercaptoethanol (40 min: 37°C) and the product purified by chromatography on a Waters Sep-Pak C 18 cartridge as describ. 4 for 4 the reductior of peptide in Example 1. After rotary evaporation, the sample was reacted with SPDP labelled RAT 1C3/86, which had been labelled with 5 propyldithiopyridine groups/antlbody as described in I xFvample 1 (16h: 25 0 Finally, the digoxin/antibody conjugate was purified by gel filtration chromatography on Superose 12, EXAMPLE 5 Preparationof HIV peptide/anti-glycophorin F(ab) conjwate An alternative reagent for the detection of anti-HIV antibodies uses an F(ab) 2 derivative of the anti-glycophorin antibody, RAT 1C3/86 (2 mg/mi in 70mM acetate 100mM sodium chloride pH 3.5) was digested with 10 pg/ml pepsin (Sigma P6887) for 40 min at 37 0 C and the reaction terminated by the addition of 1/10 vol 1.5M Tris base. After -21-, overnight dialysis into a buffer containing 5mM sodium phosphate pH the antibody fragment was purified by ion-exchange chromatography on DEAE cellulose on a 5-300mM gradient of sodium phosphate pH SPDP labelling of the F(ab)'2 fragment, reduction of the poptide 3.2, conjugation of peptide 3.2 to F(ab)'2 RAT 1C3/86, purification of the peptide conjugate, preparation of reagent for assay and testing procedure were tarried out as described for 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-gvcophorin Fab' conjugate Another alternative reagent employs a univalent Fab' fragment as the

EBM.

F(ab) 2 RAT 1C3/86 in phosphate buffered saline was incubated with lOmM mercaptoethanol for lh at room temperature. Then 15mM lodoacetamide 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.

SPDP labelling of the s-carboxymethylated Fab', reduction of the peptide 3.2, conjugation of peptide 3.2 to the Fab'-TNB (thionitrcbenzoyl) AT fragment of RAT 1C3/86, purification of the peptide conjugate, preparation of reagent for assay and testing pro edure were carried out as described for the whole antibody conjugate.

EXAMPLE 7 Preparation of Melittin as alternative ESM A peptide from the bee venom, melittin (CVLTTGLPALISWIKRKRQQ), 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-Blotin linkage to couple EBM and ABM The ABM and EBM need not be covalent'y coupled, One alternative is avidin-blotin linkage, Preparation of Btotin-labelled melittin The melittin peptide (10 mg) was reduced with mercaptoethanol as described for the 3.2 peptide in Example 1. After rotary evaporation, the

A

ADC/295c -22sample was resuspended in 2 ml 0.1M fris-HCl, 5mM 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 the biotinylated derivative separated from byproducts on a Sephadex 310 column.

Preparation of Avidin-labelled peptide 3.2 The 3.2 peptide is coupled to avidin in the same manner as it was coupled to antibody in Example 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.

S 4 ADC/295c -23- 1

Claims

1. An agglutination reagent For direct detection of an analyte in a blood sample, comprising an erythrocyte binding mol.cule 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 reagent which comprises a conjugate comprising at least one erythrocyte binding molecule conjugated with ai 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 ncn-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. The reagent of claim 4 further characterised in that the reagent is prepared by forming a heterobifunctional hybrid of a homobifunctonal erythrocyte-binding antibody and a homobifunctional analyte-binding antibody, said reagent comprising a detectable amount of Shomobifunctional erythrocyte-binding antibody, wherein said homobifunctional antibody does not auto-agglutinate erythrocytes. B/0005e r I- 25

6. The reagent of any one of claims 1 to 3 wherein said eryth,'ocyte 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. HB9893 or an F(ab) 2 or Fab' fragment of said antibody. 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 bindin 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, ar'; i not derived from an antibody or lectin. The 'eagent according to claim 1 wherein the attachment is by means of covalent coupling.

16. The reagent according to any one of claims 1 to 15 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 to 16, further comprising a secondary antibody or specific binding fragment thereof 4p, which specifically recognizes an overlapping epitope formed by the S/ inding of said analyte binding molecule to said analyte. /0005e 26

18. The reagent of claim 17, wherein said secondary antibody is provided in unconjugated form.

19. The reagent of claim 17, 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 mellitin which does not by itself agglutinate erythrocytes.

22. The reagent according to any one of claims 1 to 21, wherein the analyte is D-dimer or crosslinked fibrin degradation products. 23, The reagent according to any one of claims 1 to 21, wherein the analyte is an antibody to HIV.

24. The reagent according to any one of claims 1-3, 6-14, 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 claim 24 wherein the HIV-1 peptide is gp 41 peptide as hereinbefore defined.

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. 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, L 4-16, 21-31 and observing whether the erythrocytes are agglutinated and S correlating the agglutination with the amount of analyte present in the S sample. VIrPt4 CLB/0005e 27

33. A direct agglutination assay for the presence or amount of an analyte in a whole blood sample from a subject whicii comprises forming a mixture of sample, erythrocytes, and an agglutination reagent according i to claim 3; observing whether the erythrocytes are agglutinated and directly correlating the agglutination with the amount of analyte present. i 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 erythrocyte 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. A direct agglutination assay 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 multivalent monoclonal antibody which binds to erythrocyte membranes or a binding fragment thereof, and 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 agglutination 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 analy+e binding molecule to the analyte, and correlating the presence or degree of agglutination with the presence or quantity of the analyte in C sample. C C/0005e r 1.11 28

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 orythrocyte binding molecule. 39, A direct agglutination assay for the presence or amount of an analyie in a sample which comprises forming a mixture of a sample, erythrocytes, and an agglutination reagent which comprises a iconjugate 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 anafyte 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 detectable amount of homobifunctional erythrocyte-binding antibody, but said homobifunctional erythrocyte-binding antibody essentially does not auto-agglutinate S. 25 erythrocytes. The assay of claim 32 wherein the analyte is an antigen or hapten and the analyte binding molecule is an antibody.

41. The assay of claim 32 wherein the analyte is an antibody and the analyte binding molecule is an antigen or hapten recognized by said antibody.

42. The assay of claim 32 wherein the analyte is an antibody and the analyte binding molecule Is an antl-idlotypic antibody.

43. The assay of any one of claims 32 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. S44. The assay of any one of claims 32 to 43, wherein the analyte lacks repeating epitopes. B/0005e 29 The assay of claim 37 or 38, wherein the secondary binding molecule is also conjugated to an erythrocyte binding molecule.

46. 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.

47. 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,

48. 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,

49. The reagent of claim 48 in which the antibody fragment is non-univalent.

50. The reagent of claim 47, wherein the erythrocyte binding S molecule is a peptide having an affinity for the erythrocyte membrane but is incapable of lysing erythrocytes. 51, The reagent of claim 47, 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.

52. The reagent of claim 50, wherein the peptide is a nonlytic, erythrocyte binding fragment which does not by itself agglutinate erythrocytes. 30

53. 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.

54. The reagent according to any one of claims 47 to 53, wherein the analyte is an antibody to HIV. The reagent of claim 47, wherein said erythrocyte binding molecule is an antibody or specific binding fragment thereof,

56. The reagent according to claim 55, 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.

57. The reagent according to claim 47, wherein the erythrocyte binding molecule corresponds to a fragment of a lectin, said fragment having only one erythrocyte binding site.

58. The reagent of claim 47, wherein said erythrocyte binding molecule comprises a univalent fragment derived from an anti-erythro;yte antibody and said analyte binding molecule comprises a univalent fragment derived from an anti-analyte antibody.

59. The reagent according to claim 47 or 48, 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. The reagent according to claim 13 or 59, wherein the antibody or F(ab) 2 or Fab' fragment thereof is raised against integral membrane S protein 1, membrane attached glycoprotein C4, integral memLbane glycoprotein, ankyrin, spectrin, glycophorin, glycolipid, glycosphingolipid, protein 4-1 or F-actin.

61. The reagent of claim 47, 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.

62. The reagent according to any one of claims 47 to 61, wherein the erythrocyte binding molecule but not the analyte binding molecule binds a surface protein or glycoprotein. ii1,. 63. The reagent according to claim 47, wherein the attachment is by means of covalent coupling. LB/0005e 31

64. The reagent of any one of claims 1-5, 47-49, wherein the erythrocyte binding molecule is an antibody for glycophorin or a specific binding fragment thereof. The reagent of claim 64, further characterized in that the erythrocyte binding molecule is an antibody for glycophorin A or a specific binding fragment thereof.

66. The reagent of claim 53, wherein the peptide is a nonlytic, erythrocyte-binding fragment which does not by itself agglutinate erytrocytes.

67. The reagent of claim 53 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.

68. The reagent of any one of claims 14, 27, 47, 51, 53, 61 or 67, in which the erythrocyte binding molecule corresponds to a nonlytic, erythrocyte binding fragment of mellitin,

69. The reagent of any one of claims 21, 66 or 68 in which the erythrocyte binding molecule corresponds to mellitin 7-26, The reagent of claim 14 or 53 wherein the peptide corresponds to a nonlytic, erythrocyte-binding, fragment of protamine which does not by itself agglutinate erythrocytes.

71. An indirect agglutination assay for the presence or amount of an analyte in a blood sample which comprises incubating the sample with a an agglutination reagent according to any one of claims 47, 48, 50 to 68 and a soluble analyte binding reagent, whereby said agglutination reagent compete' 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.

72. An indirect agglutination assay for the presence or amount of an analyte in a sample from a subject which comprises forming a mixture of sample, erythrocytes and an agglutination reagent according to claim 49, permitcing said agglutination reagent to compete with sample analyte for the analyte binding sites of the analyte binding reagent, observing whether agglutination occurs, and inversely correlating the degree of agglutination with the presence or amount of analyte present. CLB/0005e A__ rr I 32

73. An indirect agglutination assay for the presence or amount of the analyte in a sample which comprises 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, permitting said conjugate to compete with sample analyte for the analyte binding sites of the analyte binding reagent, observing whether agglutination occurs, and inversely correlating the degree of agglutination with the ar.ount of analyte present.

74. An indirect agglutination assay 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 (1) an agglutination reagent which comprises 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 erythrucytes but only in the presence of a multivalent analyte binding agent, and (11) 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, observing whether agglutination occurs, and inversely correlating the degree of agglutination with the presence or amount of analyte present. The assay of claim 74, wherein the binding fragment is non-univalent; said assay being further characterized in that at no time are the sample or the conjugala with erythrocytes exogenpus to the subject. 76, The assay of any one of claims 32-46, 71-75 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.

77. The assay of claim 34 or 73 in which the peptide is a bee venom-like peptide.

78. The assay of claira 34 or 73 In which the erythrocyte binding molecule corresponds to a nonlytic, erythrocyte-binding fragment of /K mellitin which does not by itself agglutinate erythrocytes. /0005e 7- ~ran~s~ ~i~uaan-*-un~~~ 33

79. The assay of claim 78 in wh;ch the peptide corresponds to mellitin 7-26. The assay of claim 73 in which the analyte analogue is a peptide or protein and said erythrocyte binding molecule and analyte analogue are conjugated by a simnri pe(.cide bond.

81. The assay of claim 80 in which the erythrocyte binding molecule corresponds to a nonlytic, erythrocyte-binding fragment of mellitin.

82. The assay of any one of claims 33, 72 or 75 in which the erythrocyte binding molecule is an anti-glycophorin monoclonal antibody or a specific binding fragment thereof,

83. The assay of any one of claims 33, 39, 72 or 75, wherein the erythrocyte-binding fragment is derived from an anti-glycophorin antibody.

84. The assay of claim 33 or 72, wherein the erythrocyte binding molecule is an antibody for a surface protein or glycoprotein or a specific binding fragment thereof. The assay of claim 36 or 84, wherein the erythrocyte binding molecule is an antibody for glycophorin or a specific binding fragment thereof.

86. The assay of claim 85, furtner characterized In that the erythrocyte binding molecule Is an antibody for glycophorin A or a specific binding fragment thereof,

87. The assay of claim 74, wherein the agglutination reagent comprises a conjugate of an Intact multivalent monoclona antibody which binds to erythrocyte membranes or a multivalent 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,

88. A test kit for use in direct agglutination assays which comprises 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 a secondary binding molecule which binds to a new epitope formed by the binding of the analyte binding molecule to the analyte.

89. A direct agglutination assay test kit comprising an I agglutination reagent according to any one of claims 1 to 31 and a 'reference solution containing a known quantity of analyte. A1.r p0 CLB/0005e 34 A test kit for use in direct agglutination assays which comprises an agglutination reagent according to any one of claims 1 to 16, 20-31 and a secondary antibody which binds to a new epitope formed by the binding of the analyte binding molecule to the analyte.

91. The test kit of claim 88 or 90, wherein the secondary antibody is conjugated to an erythrocyte binding molecule.

92. A test kit for use in indirect agglutination assays which comprises an agglutination reagent according to any one of claims 47 to 68 and a soluble analyte binding reagent.

93. The test kit of claim 92, further comprising a reference samnle containing a known quanti of analyte,

94. An agglutination reagent for the direct detection of an analyte in a whole blood sample, which reagent is substantially as herein described with reference to any one of the Examples.

95. A direct agglutination assay for the detection of an analyte in a whole blood sample which assay is substantially as herein described with reference to any one of Examples 1, 2, 3, 5 or 6.

96. A SPDP-conjugated digoxin/anti-glycophorin antibody conjugate substantially as herein described with reference to Example 4. DATED this TWELFTH day of MARCH 1992 Agen Limited Patent Attorneys for the Applicant N SPRUSON FERGUSON CLB/0005e