LATEX AGGLUTINATION USING AVIDIN/BIOTIN SYSTEM

Abstract: A reagent for detecting an analyte of interest in a sample fluid comprises: a) an inert particle; b) a plurality of avidin molecules noncovalently attached to the surface of the particle; and c) derivatized biotin molecules complexed with at least a portion of the avidin molecules, each such derivatized biotin molecule having the structure Formula (I), wherein A is a ligand specific for the analyte of interest and -R- is a linker group which covalently joins A to biotin.

Description: LATEX AGGLUTINATION OSIHG AVIDIH/BIO IH SYSTEM Background of the Invention The present invention relates to an agglutination test and reagent for detecting the presence of an analyte of interest, e.g., an antigen or antibody, within a sample specimen. One form of agglutination test which is currently in use, called a latex agglutination test, utilizes a suspension of plastic particles or beads carried in an aqueous vehicle. Describing a typical latex agglutination test as it would be performed for the detection of an antibody, a reagent is prepared for the latex suspension by coating the beads or particles in the suspension with ' a ligand, for example an antigen specific for the tested-for antibody. The reagent so prepared is then mixed, as on a slide, with a diluted or undiluted serum specimen. Antibody present in the serum sample or specimen attaches to the antigen coated on the beads or particles, resulting in agglutination of the beads or particles which may be visually observed, indicating a positive test. In a negative test, agglutination does not occur and the mixture of specimen and reagent remains smooth and milky in appearance. An agglutination test as just described may be quickly performed and is relatively simple to carry out, which has obvious advantages. Despite the advantages indicated above, certain disad¬ vantages have characterized prior art agglutination tests, thereby negating their general acceptance. Specifically, many latex agglutination tests require very high titered pure ligand preparations. Additionally, coating of the latex beads or particles with ligand is a difficult task when passive adsorption of the antibody is relied upon, since this depends upon the presence of charged surfaces on the latex particles. Also, functional groups present on the ligand may be determinative of whether or not the ligand will be properly adsorbed on the latex particles. Furthermore, latex particles generally adsorb ligand molecules randomly, thus rendering a portion of the ligands adsorbed on the particle use¬ less. Moreover, as in the case of antibodies, only micro amounts of antibody are adsorbed to the latex particle relative to the amount added. Accordingly, the maximum adsorption — and therefore the maximum test sensitivity — requires the use of ultrapure antibody, which is usually difficult to obtain. Additionally, impurities present in the antibody preparation may compete with the antibody for adsorption sites on the latex particle surface. If passive adsorption is not possible in producing the coating, crosslinking or bridging with chemicals such as glutaraldehyde may be used. Chemicals such as these, however, may inactivate the ligand to the extent that it may no longer function to bind another ligand, e.g., an antigen or antibody. For these and other reasons, relatively few latex agglutination tests are available for viral antibody or antigen detection. Agglutination tests are also known which rely upon cellular bodies, e.g., treated red blood cells, as the centers of cell aggregates formed in the agglutination. An assay performed with treated blood cells, however. involves problems .such as maintaining a proper supply of the processed cells , shel l-lite problems , expense , etc . U . S. Patent No . 4 , 434 , 150 (Azad et al . ) discl oses an immunological detecting reagent which comprises a water sol ubl e polymer hav ing an antibody or antigen attached via an avidin-biotin l inkage. The water soluble poly¬ mer al so has a pl ur al ity of detectable marke rs attached to i t. U.S. Patent No. 4,228,237 (Hevey et al.) discloses a method for determining the presence of a ligand in a liquid medium which utilizes a detectable enzyme com¬ plex to which is bound an antigen or antibody corre- sponding to the ligand. The binding is via the avidin- biotin system. The detectable enzyme complex is then contacted with an insoluble phase containing a specific binding substance for the ligand. The detectable en¬ zyme complex binds with the specific binding substance and is then detected in the insoluble phase. U.S. Patent No. 4,298,685 (Parikh et al.) discloses a method for the quantitative determination of a biologi¬ cal substance in a test sample which comprises mixing a test sample containing an unknown amount of antigen with known amounts of biotin labelled antibody and antigen labelled with detectable enzyme. The enzyme labelled antigen competes with the unknown amount of free antigen in the system for the biotin labelled antibody. Two different antigen-antibody complexes result: one labelled with both enzyme and biotin and the second labelled only with biotin. The antigen- antibody complexes are then contacted with an insoluble solid support coated with avidin. The avidin binds with the biotin labelled complexes to form a precipitate comprising solid support-avidin-biotin-antibody-anti- gen. Enzyme activity is then measured in order to quantitate the biological substance. U.S. Patent Nos. Re. 31,712 and 4,478,914 (Giese) disclose methods for coating the surface of beads, particles, spheres etc. with biotin or caproylamidobiotin N-hydroxysuccinimide to form a multi-layered product having successive, repetitive layers of avidin-extender attached to the biotin. U.S. Patent Nos. 4,141,965 and 4,210,622 (Soothill et al.) and U.S. Patent No. 4,331,649 (Chantler et al.) each disclose the estimation of an immune complex using a latex particle agglutination test. The latex particles are coated with a constituent of the immune complex and the constituent is attached to the latex bead via a coupling agent such as dinitrophenol. Next, the coated latex beads are contacted with a sample containing an unknown amount of an antibody to the constituent. Immune complex is then measured by the inhibition of the agglutination reaction. U.S. Patent No. 4,582,810 (Rosenstein) describes a diagnostic particle for use in an aqueous suspension in agglutination tests. The particle comprises a carboxylated latex particle having avidin molecules linked thereto through covalent amide bonds. Biotin molecules are complexed with the avidin molecules and antibody molecules are linked through covalent amide bonds to a significant proportion of the biotin mole¬ cules. Thus, the latex particle is coated with anti¬ body and is useful for detecting an antigen specific for the antibody. However, a disadvantage of such a diagnostic particle is that the formation of the covalent amide bonds between the latex particle and the avidin molecules requires additional steps which add to the time and expense of preparing such a particle and also introduces the possibility of deactivating antibody binding site(s). None of the above-mentioned references disclose an immunoassay agglutination test which utilizes non- soluble particles coated with one or more avidin- biotin-spacer arm-ligand complexes. A general object of the invention is to provide an improved agglutination test which obviates many of the problems and disadvantages associated with such tests as currently available. Summary of the Invention A reagent for detecting an analyte of interest in a sample fluid comprises: a. an inert particle; b. a plurality of avidin molecules noncovalently attached to the surface of the particle; and c. derivatized biotin molecules complexed with at least a portion of the avidin molecules, each such derivatized biotin molecule having the structure: wherein A is a ligand specific for the analyte of interest and -R- is a linker group which covalently joins A to biotin. The present invention also provides a reagent for de¬ tecting an analyte of interest in a sample which com¬ prises: a. an inert pa ticle; b. a plurality of avidin molecules noncovalently attached to the surface of the molecule; and c. derivatized biotin hydrazide molecules complexed with at least a portion of the avidin molecules, each such derivatized biotin hydrazide molecule being covalently bound to a carbohydrate-containing ligand specific for the analyte of interest. 10 15 20 25 30 35 Detailed Description of the Invention The present invention provides a reagent for detecting an analyte of interest in a sample, which comprises: a. an inert particle; b. a plurality of avidin molecules noncovalently attached to the surface of the particle; and c. derivatized biotin molecules complexed with at least a portion of the avidin molecules, each such derivatized biotin molecule having the structure: wherein A is a ligand specific for the analyte of interest and -R- is a linker group which covalently joins A to biotin. -R- may be represented by the structure: O II C-(CH 2 ) n — H wherein n is an integer between 2 and about 14. -R- may also be represented by the structure: H I = N - N - wherein A comprises a carbohydrate and -R- is covalently linked to A by means of the double bond of -R-. In a preferred embodiment, the derivatized biotin molecule has the following structure: The particle may be polyacrylate, polystyrene or polycarbonate. The particle may also be any other inert particle which is capable of being suspended in a liquid vehicle, e.g. water, such as an inorganic clay particle, e.g. bentonite, or a fixed or tanned red blood cell. In the reagent of the present invention the avidin molecules may be passively adsorbed to the particle surface and at least 1 and up to 4 derivatized biotin molecules may be complexed with each avidin molecule. In one embodiment of the invention, the analyte of interest is an antibody or an antigen. The antibody to be detected may be specific for a virus, e.g. herpes simplex virus or human immunodeficiency virus. In another embodiment of the invention, the analyte of interest is a hormone. In still another embodiment of the invention, the ligand comprises an antibody, an antigen, or a hapten. Furthermore, the antibody may be a monoclonal antibody. Moreover, the antigen may be a polysaccharide or a glycoprotein. Another reagent for detecting an analyte of interest in a sample fluid comprises: a. an inert particle; b. a plurality of avidin molecules noncovalently attached to the surface of the particle; and c. derivatized biotin hydrazide molecules complexed with at least a portion of the avidin molecules, each such derivatized biotin hydrazide molecule being covalently bound to a carbohydrate-containing ligand specific for the analyte of interest. In the reagent utilizing biotin hydrazide, the ligand may comprise a polysaccharide or a glycoprotein. The carbohydrate groups of the ligand must be pretreated so as to form aldehyde groups which react with the biotin hydrazide. The particle may be polyacrylate, polystyrene or polycarbonate. The avidin molecules are passively adsorbed to the particle surface. Additionally, the analyte of interest may be specific for a virus, e.g. herpes simplex virus or human immunodeficiency virus. Moreover, the analyte of interest may be a hormone. Also provided are liquid suspensions comprising the reagents of the present invention in a suitable liquid medium, e.g., water. Also provided is a method for detecting the presence of an analyte of interest in a sample which comprises contacting the sample with a reagent of the present invention, wherein the reagent comprises a ligand specific for the analyte of interest, so as to form a complex between the analyte of interest and the reagent, wherein the complex is in the form of a precipitate or agglutinate, and detecting- the presence of the complex and thereby the presence of the analyte of interest. The contacting may be effected on the surface of a glass slide and may be done manually or mechanically. The precipitate or agglutinate may be detected visually, microscopically or photometrically. The present invention provides an agglutination test which utilizes a suspension of solid particles as the core material in the aggregates that form during the agglutination procedure, and an avidin-biotin linking system employed for coating such particles with a ligand. Such particles react and combine with the analyte of interest, which results in an agglutination indicative of a positive test. According to the present invention, solid inert particles, as exempli¬ fied by the plastic beads or particles that typify a latex suspension, are coated with a ligand through an avidin to biotin bond, wherein the avidin is attached to the particle surface. The particles and the antigen may be subjected to a relatively gentle nondestructive pretreat ent, after which their attachment is readily accomplished. The attachment through the avidin-biotin bond is simple and specific. Since no harsh chemicals are used in the adsorption procedure, it is contem¬ plated that almost any ligand may be bound to the surfaces of the particles in the suspension. Because of the strength and specificity of the bond, relatively low concentrations of the ligand are required for adsorption on the particles of the suspension. More specifically, the present invention provides that the particles of the suspension are ' coated with avidin, which is readily adsorbed onto the particle surfaces. Avidin, as is known in the art, has four binding sites for biotin. Thus, avidin-coated particles have poten¬ tial binding sites for a biotinylated antigen which is four times the number of avidin proteins attached to the particle surfaces. The ligand employed in ag¬ glutination tests may be easily and nondestructively conjugated with biotin. Mixture and interaction of a biotinylated ligand and suspension of avidin-coated particles readily produces a reagent suspension for the determination, in an agglutination test, of the pres¬ ence or absence of an analyte of interest specific for the ligand. Avidin is a glycoprotein of approximately 68,000 molec¬ ular weight present in egg white, which has been found to have an extraordinary high affinity for the small molecular weight biotin. Thus, biotin and avidin in¬ teract specifically and under mild conditions to form a strong, stable interlinked complex. Avidin has four binding sites, each of which may bind to a biotin mole- cule and this binding persists when avidin attaches to a structure such as the surface of an inert particle or bead. Certain analogs or derivatives of avidin suggest their use as a substitute for avidin. A typical exam¬ ple of an avidin analog is streptavidin, which appears to be a 60,000 molecular weight protein and like avidin has four binding sites for biotin and a binding affini¬ ty similar to its analog. Exemplifying avidin deriv¬ atives applicable to the invention are such materials as succinyl avidin, ferritin avidin, and crosslinked avidin. As used hereinafter, the term "avidin compo- nent" refers to avidin as well as its recognized ana¬ logs and derivatives. Avidin is readily available in highly purified form and readily attaches to the sur¬ face of an inert particle or bead. Biotin, also known as Vitamin H, has a molecular weight of about 274. Biotin easily conjugates with many proteinaceous materials without altering the biological activity of the protein. Thus, it is easily conjugated with an antigen. Biotin derivatives and analogs are contemplated for use in the present invention. Examples of such materials are biocytin, biotin hydrazide, biotinyl-N- hydroxysucciniraide ester, and other long chain biotinyl molecules. As used herein, the term "biotin component" refers to biotin as well we its recognized analogs and derivatives. Biotin hydrazide is a particularly useful biotin deriv- ative contemplated for use in the present invention in that it may be conjugated to carbohydrate residues present in polysaccharides or glycoproteins. To achieve this conjugation the carbohydrate residues are oxidized with periodate to form alpha-hydroxy aldehyde groups which cross-link with the free amino groups of the biotin hydrazide. The reaction generally proceeds as follows: 0 ( 1 Carbohydr ate— C + HN=N Biotin f Ca rbohydrate— — C=N N Biotin I f H H Biotin hydrazide conjugation may be achieved using the method of D. 0. Shannessy et al., Immunol. Letters _[: 243-277 (1984) . For example, to biotinylate a carbohy¬ drate containing antigen, the concentration of the antigen is adjusted to about 1.0-2.0 mg/ml in 0.1M sodium acetate, pH 5.5. Next, 0.01M sodium periodate is added to the antigen solution and the mixture is incubated in a bath at 0°C for 20 minutes. Glycerol, 0.015M at 0°C, is added for 5 minutes to stop the reac¬ tion. The solution is then dialyzed overnight at room temperature against sodium acetate, pH 5.5. Biotin hydrazide is added to a concentration of 0.01M and then the solution is incubated with gentle shaking for two hours at room temperature. The solution is then dia¬ lyzed against phosphate buffered saline, 0.01M, pH 7.4, overnight at room temperature. The biotinylated antigen is then recovered and stored at 4°C. The attachment of a ligand specific for an analyte of interest to the particles of the suspension is achieved by coating the particles of the suspension with avidin, preparing a biotinylated ligand and reacting a mixture of the two to produce a suspension where the ligand is attached to the surfaces of the particles through an avidin-biotin link. Since avidin has four binding sites, such a method produces particles having a multiplicity of ligand molecules attached. The resulting particle is useful as a reagent for de¬ tecting the presence of an analyte of interest. Another system contemplated comprises particles in suspension which have a biotin coating, a biotinylated ligand bound to the particle surfaces through avidin which bridges the biotin on the particle surfaces and the biotin being conjugated with the ligand. Suspensions for carrying out the agglutination test of the present invention include the so-called latex sus¬ pensions which are suspensions of inert plastic parti¬ cles or beads carried in a liquid vehicle, such as water. The beads may be of various plastic composi¬ tions, such as a polyacrylic resin, a polystyrene resin or a polycarbonate resin. In the usual instance, the beads or particles will have diameters within the range of 0.5 microns to 3.0 microns. Other contemplated suspensions are suspensions of inert particles, such as inorganic clays, as exemplified by bentonite suspen¬ sions, and fixed or tanned red blood cells. A bentonite suspension may be prepared as a suspension of 0.5 g of bentonite in 500 ml distilled water and then centrifuged at 150 x g for 10 minutes. The sediment is discarded and the supernatant recentrifuged at 1400 x g for 10 minutes. This sediment is resuspended in 100 ml PBS (pH 7.4) solution in a 250-ml Erlenmeyer flask, autoclaved, and refrigerated as "stock" bentonite. Immediately before coating them with avidin, the particles' are collected from a 10-ml suspension of stock bentonite and centrifuged at 1400 x g for 10 minutes. The supernatant is discarded and the bentonite sediment resuspended in 2 to 3 ml 0.05 M PBS that contains avidin (1 mg/ l) . The avidin bentonite suspension is permitted to stand at room temperature with occasional mixing on a Vortex stirrer over a period of 30 minutes. The coated particles are sedimented by centrifugation of the suspension at 1500 x g for 15 minutes and then resuspended in PBS (pH 7.4) . Biotinylated ligand may then be added to coat particles with the ligand and the resulting particles used in the same manner as the latex beads in an agglutination assay. Tanned red cells may be coated with ligand by the following method. Tanned erythrocytes are prepared from sheep blood collected in sterile 3.8 sodium citrate or Alsever solution. Blood is aged for 1 to 8 weeks at 4°C and then washed three times with PBS (pH 7.2). During the washing procedure, the buffy coat is removed by aspiration, and the remaining packed eryth¬ rocytes are resuspended to make a 2.5% concentration. The optimal tannic acid (reagent grade) dilution may vary from 1:20,000 to 1:160,000 (wt/vol) and should be determined for each batch of cells. The tannic acid is diluted in PBS (pH 7.2) within one hour of use. Equal volumes of the 2.5% erythrocyte suspension and the tannic acid solution are mixed and incubated in 37°C water bath for 20 minutes. The cells are centrifuged at 500 x g for 10 minutes and washed once with PBS (pH 7.2). They are then resuspended to a 2.5% concentra¬ tion in PBS (pH 6.7) . For sensitization of the cells with avidin, the avidin is diluted in PBS (pH 6.7) and 1 volume of the optimal avidin dilution (0.1 mg/ml) and 1 volume-of 2.5% tanned cells are mixed and incubated for 30 minutes at 37°C. The cells are centrifuged at 500 x g for 10 minutes, washed twice with a 1% normal swine serum in PBS (pH 6.7) diluent. Biotinylated ligand, prepared as previously described, may then be used to coat the red cells. The coated red cells are washed and then used in the same manner as the latex beads in an agglutination assay. An agglutination test according to the present inven¬ tion is conveniently carried out as in a standard slide agglutination assay. Thus, a reagent comprising a suspension of particles of the present invention is mixed on a slide with a deposit of diluted or undiluted serum specimen. Mixing is accomplished by hand-tilting or mechanical rotation of the slide. The latex suspen¬ sion should not be permitted to dry, and if necessary, the slide may be kept covered to prevent evaporation. Reaction times may vary but, in the usual instance, are relatively short, i.e., under ten minutes. In a positive test, the particles of the suspension visibly agglutinate. In a negative test, the mixture on the slide does not agglutinate and appears smooth and milky. Example 1 Describing a specific example of how the invention may be carried out, a latex suspension comprising polycarbonate beads of 0.7 micron diameter coated with avidin was first prepared. The suspension was prepared by incubating a mixture of polycarbonate beads suspended in TRIS (2-amino-2- hydroxymethyl -1, 3-propanediol) buffered aqueous sa¬ line solution (pH 8.6) containing 0.02 percent by weight avidin at room temperature for two hours. The particle concentration in the saline solution was 5 g per 100 ml of solution. The coated particles that resulted were centrifuged and resuspended in TRIS buff¬ ered saline solution (pH 7.6) to produce a suspension containing coated particles and a particle concentra¬ tion of 5 g per 100 ml ^ of solution. The resulting particles had avidin bound thereto via passive adsorp¬ tion, i.e. hydrophilic or electrostatic interactions. The use of passive adsorption is faster, less expensive and less likely to inactivate the binding siteu of the avidin attached to the particle than if the avidin was covalently bound to the particle. The ligand, i.e., antigen, utilized was a cell lysate of herpes simplex viral particles. The antigen was used as • a crude preparation of viral particles. The ability to use relatively impure antigen is another advantage of the present invention. Latex particles having passive attachment of the antigen directly to the latex bead, would result in an attachment that was weak and therefore the antigen would be required to be highly purified in order to optimize binding. Antigen specific for herpes simplex virus antibody was biotinylated using standard procedures. Thus, a mix¬ ture of antigen and phosphate buffered saline solution was prepared (20 mg antigen per 1 ml solution) . Biotin ester (biotin-N-hydroxysuccinimide) was dissolved in dimethylformamide (50 mg ester per 1 ml solvent) . A mixtur e was prepared f r om 0 .125 ml anti gen sol ution , 0 .02 ml sodi um bica rbona te ( 0 .5 NaHC0 3 , pH 9 .0 ) , 0 .04 ml wate r , and 0 .01 ml biotin este r sol ution and the mixtur e was incubated one hour at room temperatur e. After incubation, 0.025 ml of 1.0 M NH C1 was added to stop further reaction. The mixture was then dialyzed against phosphate buffered saline solution. The biotinylated herpes simplex antigen was then isolated. This biotinylation procedure is mild and preserves the activity of the antigen. The biotinylated antigen was then mixed with the latex suspension containing avidin-coated beads using equal amounts of the antigen and the latex suspension. The mixture was reacted on a vibrator at room temperature for ten minutes to produce a reagent useful in an ag¬ glutination test to detect the presence of herpes sim¬ plex virus antibody in a serum sample. In performing a slide test, ten microliters of latex particle reagent, prepared as above, was mixed on a slide with 40 to 60 microliters of a diluted serum specimen. The slide was then hand tilted to mix the components. A positive test exhibiting agglutination ordinarily occurred in approximately eight minutes. The test set forth above was performed with different serum samples. Each sample was assayed using an indi¬ rect hemagglutination titer performed in a conventional manner and as known in the art. Each sample was also assayed utilizing the avidin-biotin enhanced latex agglutination procedure described above. Table 1 sets forth the results obtained. In the column entitled "Avidin-Biotin Enhanced Latex Agglutination", a "+" or "-" is indicative of a positive or negative result, respectively. In the column entitled "Hemagglutination Titer", the number indicated is the reciprocal of the highest serum dilution that had antibody. TABLE 1 Heroes Simplex Virus Antibody Assay Clinical Indirect Avidin-biotin Samples Hemagglutination enhanced latex - Tϋ≤E . agglutination 1 < 8 2 32 3 8 4 128 + 5 128 + 6 312 + 7 32 + 8 < 8 9 < 8 + 10 256 + 11 65 + 12 512 + 13 < 8 + 14 8 + 15 32 + 16 < 8 17 512 + 18 8 + 19 64 + 20 64 + 21 32 + 22 < 8 23 < 8 24 < 8 25 2048 As demonstrated above, the latex agglutination test of the present invention, with agglutination enhanced by an avidin-biotin linkage of the antigen to the parti¬ cles of the latex suspension, prov.ides a reliable meth¬ od for determining the presence or absence of an anti¬ body in the specimen or sample tested. The test is rapidly performed and carried out far more easily than is the indirect hemagglutination test with which the agglutination test of the invention was compared. EXχbU£L£-2. The procedure used was identical to that of Example 1 except that an activated biotin ester was used, namely N-biotinyl-6-aminocaproic acid N-hydroxy succinimide ester. This biocin ester is commercially prepared as ENZOTIN m (Enzo Laboratories, New York, New York) and is a long chain activated biotinyl molecule having the following structure: o // HN NH 0 N-O-C- ( CH 2 ) s -NH-C-(CH,) 4 ' « w O The N-biotinyl-6-aminocaproic acid N-hydroxy succinimide ester used may be prepared according to the method of Costello, S.M. et al . (Clin. Chem. __[ ( 9 ) : 1572-1580 (1979)) . The long chain biotinyl molecule has a spacer arm comprising seven atoms, which is the chain comprising tne six carDons and one nitrogen group. It is contemplated that a spacer arm may be prepared which has an amino terminal end attached to a chain of from two to fourteen carbon molecules by modi¬ fying the procedure of Costello et al. The present invention contemplates the use of long chain biotinyl molecules which have spacer arms having from two to fourteen carbon molecules. Reaction of the long chain biotinyl molecule with the antigen results in the antigen being attached to the activated long chain biotinyl molecule via an amide bond formed between the free amino groups of iysine or arginine, or any primary amino group, in the antigen and the activated biotinyl molecule. The use of a long chain biotinyl molecule decreases the steric hindrance of the antigen molecules on the surface of the particle or bead and therefore provides a more sensitive assay. EXAMPLE 3 The linker arm (N-biotinyl-6-aminocaproic acid) may also be applied to the detection of antigens in biological fluids by binding specific antibodies to biotin via the linker arm. This biotin: linker arm: antibody complex may than be contacted with the avidin coated latex bead as described earlier to create a bead with active antibody. Applicants have detected Hepatitis B surface antigens in serum samples in excellent agreement with other accepted methodologies. Monoclonal antibody to Hepatitis B surface antigens was obtained from Hybritech, Inc. Hepatitis B surface antigens, formerly known as Australian antigen, have antigenic determinants which permit their classification into various sub-types. One antigenic determinant common to all Hepatitis B antigens is termed A and two mutually exclusive determinants are called D and Y. The monoclonal antibody used in these studies was produced against the A:D combination. The antibody protein was conjugated to the N-biotinyl- 6-aminocaproic acid N-hydroxy succinimide obtained from Enzo Laboratories as ENZOTIN* as previously described. The biotinylated monoclonal antibody was then mixed with the latex suspension containing avidin coated beads prepared as previously described. Equal amounts of ligand and latex suspensions were used. The mixture was allowed to react on a vibrator at room temperature for 10 minutes to produce a reagent usable in an agglutination test to determine the presence of Hepatitis B antigen in various sera. To test specificity, blood serum was drawn from 10 healthy laboratory personnel known to be negative for Hepatitis B. The disclosed latex agglutination test as described found all 10 to be negative. To test sensitivity, an additional group of sera known to be positive were tested. These were obtained from the Lynchburg Hepatitis Study carried out by the National Institute of Neurological and Communicative Disease in 1976. Sera were diluted 1:2 and those found negative were retested undiluted. Results of testing this panel are shown in the Table II compared with the results of complement fixation tests for the antigen. LYNCHBURG HEPATITIS SAMPLES (HEPATITIS B) Patient * ςf T ec frex itef 1?2 1 1. 8 2 1 16 3 1: 16 4 1 16 5 1: 32 + 6 1: 32 + 7 1 32 + 8 1: 32 + 9 1 64 + 10 1: 64 + 11 1: 64 + 12 1: 64 + 13 1; 128 + 14 1: 128 + 15 1: 128 + 16 > 1:128 + 17 >1:128 + 18 *1:128 + 19 128 + 20 256 21 256 + 22 256 + 23 512 24 512 25 1:2048 + 26 1:2048 +

Claims: Whaf is claimed is: 1. A reagent for detecting an analyte of interest in a sample fluid which comprises: a. an inert particle; b. a plurality of avidin molecules noncovalently attached to the surface of the particle; and c. derivatized biotin molecules complexed with at least a portion of the avidin molecules each such derivatized biotin molecule having the structure: wherein A is a ligand specific for the analyte of interest and -R- is a linker group which covalently joins A to biotin. 2. The reagent of claim 1, wherein -R- is O II C-.CH j ^—NH and wherein n is an integer between 2 and about 14. 3. The reagent of claim 1, wherein -R- is = N-N A comprises " a carbohydrate and -R- is covalently linked to A by means of the double bond of -R-. 4. The reagent of claim 1, wherein the sample is a biological fluid. 5. The reagent of claim 4, wherein the biological fluid comprises blood, serum, or urine. 6. The reagent of claim 1, wherein the particle com- prises polyacrylate, polystyrene or polycarbonate. 7. The reagent of claim 1, wherein the particle com¬ prises an inorganic clay particle. 8. The reagent of claim 7, wherein the particle com¬ prises bentonite. 9. The reagent of claim 1 wherein the particle com¬ prises a fixed or tanned red blood cell. 10. The reagent of claim 1, wherein the avidin mole¬ cules are passively adsorbed to tne surface of the particle. 11. The reagent of claim 1, wherein at least 1 and up to 4 derivatized oiotin molecules are complexed with each avidin molecule. 12. The reagent 'of claim 1, wherein the derivatized biotin molecule has the structure: 13. The reagent of claim 1, wherein the analyte of interest is an antibody or an antigen. 14. The reagent of claim 13, wherein _ the analyte of interest is an antibody. 15. The reagent of claim 14, wherein the antibody is specific for a virus. 16. The reagent of claim 15, wherein the virus is her¬ pes simplex virus. 17. The reagent of claim 15, wherein the virus is human immunodeficiency virus. 18. The reagent of claim 1, wherein the analyte of interest is a hormone. 19» The reagent of claim 1, wherein the ligand com¬ prises an antioody, antigen, or hapten. 20. The reagent of claim 19, wherein the ligand is an antibody. 21. The reagent of claim 20, wherein the antibody is a monoclonal antibody. 22. The reagent of claim 19, wherein the ligand is an antigen. 23. The reagent of claim 22, wherein the antigen is a polysaccharide or a glycoprotein. 24. A reagent for detecting an analyte of interest in a sample fluid which comprises: a. an inert particle; b. a plurality of avidin molecules noncovalently attached to the surface of the particle; and c. derivatized biotin hydrazide molecules complexed with at least a portion of the avidin molecules, each such derivatized biotin hydrazide molecule being covalently bound to a carbohydrate- containing ligand specific for the analyte of interest. 25. The reagent of claim 24, wherein the ligand com¬ prises an antibody, antigen, or hapten. 26. The reagent of claim 25, wherein the ligand is an antibody. 27. The reagent of claim 26, wherein the antibody is a monoclonal ancioody. 28. The reagent, of claim 25, wherein the ligand is an antigen. 29. The reagent of claim 28, wherein the antigen is a polysaccharide or a glycoprotein. 30. The reagent of cl aim 24 , wherein the carbohydrate ligand is covalently bound to the biotin hydrazide by means of a carbon-nitrogen double bond. 31. The reagent of claim 24 , wherein the particle com¬ prises polyacrylate, polystyrene or polycarbonate. 32. The reagent of claim 24 , wherein the avidin mole¬ cules are passively adsorbed to the particle sur- face . 33. The reagent of claim 24, wherein the analyte of interest is an antibody or an antigen. 34. The reagent of claim 33, wherein the analyte of interest is an antibody. 35. The reagent of claim 34, wherein the antibody is specific for a virus. 36. The reagent of claim 35, wherein the virus is her¬ pes simplex virus. 37. The reagent of claim 35, wherein the virus is human immunodefiency virus. 38. The reagent of claim 24, wherein the analyte of interest is a hormone. 39. A liquid suspension comprising the reagent of claim 1 suspended in a suitable liquid medium. 40. A liquid suspension comprising the reagent of claim 24 suspended in a suitable liquid medium. 41. The suspension of claim 39 or claim 40 wherein the liquid medium is water. 42. A method for detecting the presence of an analyte of interest in a sample which comprises contacting the sample with the reagent of claim 1 or claim 24, wherein the reagent comprises a ligand specific for the analyte of interest so as to form a complex between the analyte of interest and the reagent, wherein the complex is in the form of a precipitate or agglutinate, and detecting the presence of ' the complex and thereby the presence of the analyte of interest. 43. The method of claim 42, wherein the contacting is effected on the surface of a glass slide. 44. The method of claim 42, wherein the precipitate or agglutinate is detected visually, microscopically or photometrically.

Inventors: FUCCILLO DAVID A, SHEKARCHI ISABEL C, FUCCILLO, DAVID, A., SHEKARCHI, ISABEL, C.
Applicants: MICROBIOLOGICAL ASS INC, MICROBIOLOGICAL ASSOCIATES, INC.
Application year: 1987
Main classification: G01N33
IPC codes: G01N33