WO1999064447A1 - Solution-phase elisa - Google Patents

Abstract

A method is disclosed for quantitation of an anlyte in a test solution, wherein the analyte has a specific binding affinity for a detector molecule. The method comprises the steps of attaching a target molecule to the detector molecule via a spacer; contacting the detector-spacer-target molecule complex with the test solution containing the analyte to form a binding mixture; contacting the binding mixture with a solid matrix precoated with capture molecule, which can specifically bind to target molecules; removing unbound materials; and measuring bound materials. Also disclosed is a use of the method of the present invention for the early detection of Insulin Dependent Diabetes Mellitus.

Description

SOLUTION-PHASE ELISA

Field of the Invention

This invention relates to the field of detection and quantitation of analytes . More particularly, this invention provides a simple and sensitive enzyme immunoassay based technique for the detection of analytes, wherein the analyte is allowed to react in a solution phase with a detectable molecule having a specific affinity for the analyte. This invention also provides a method for the early detection of Insulin Dependent Diabetes Mellitus (IDDM) .

Background of the Invention

The presence of bindable molecules like antibodies and receptors is most often detected by methods based on their affinity for certain molecules. For example, specific antibodies are detected by their ability to bind to their antigenic epitopes . Similarly, receptors can be detected by their ability to bind to their ligands or mimics thereof. Conversely, antigens and ligands are detected by their ability to bind to specific antibodies or receptors .

In quantitative affinity assays, bindable reactants are allowed to react with each other under conditions that facilitate binding. Following removal of unbound material, bound complexes are detected by means of a detection system. The detection system generally comprises a molecule that can bind to one of the components of the bound complex and further has an attached detectable agent, for example, a radioisotope, a fluorescent group, or an enzyme that facilitates detection .

A critical step in the assay of affinity molecules is the separation of bound and free materials. For easy separation of the bound and free materials, two general approaches have been adopted. In the first approach, one of the bindable reactants is bound to a solid matrix and thus, unbound materials can be removed by repeated washing and isolation of the solid matrix bound materials. In the other approach, the molecules are allowed to react in a solution phase and then the bound complexes are separated by further binding to materials that can be separated based on the mass of the bound complexes (e.g. immunoprecipitation) . Examples of detection techniques in the first category include the enzyme-linked immunoassays and the antibody sandwich assay. Examples of the second category include radioimmunoassays and fluorescence immunoassays.

The enzyme-linked immunoassays involve enzyme labeling of the test molecule, generally an antigen or antibody, either directly or indirectly by labeling immune complexes which bind specifically to the test antigen or antibody. The enzyme-linked immuno complexes are reacted with a substrate and the enzyme activity is monitored. Enzyme linked immunosorbent assay (ELISA) is a modification of the enzyme immunoassays where assay reactants are adsorbed onto a solid support which provides for easy separation of the bound complexes from the unbound material and excess reagents. A large number of solid support materials have been used previously. These include beads housed in a plunger coated with an antibody to form a solid-phase { U. S .

Pa tent No . 4 , 424 , 279) ; and polymeric film coated with an antibody ( U. S . Pa ten t No . 3 , 999 , 948) ; Other commonly used substrates include microtitre plates, plastic tubes, latex and glass particles, cellulose and glass fiber filters.

A modification of the enzyme-linked immunoassay technique is the "Sandwich" technique. Antibodies specific to the test, antigen are first adsorbed in excess amount onto a solid surface such as plastic well or tube. The test solution containing the antigen is then added, allowing the antigen to bind to the adsorbed antibody. Following washing of unbound antigen, an enzyme- linked second antibody is added which reacts with specific sites on the bound antigen. The second antibody is added in excess to ensure binding of all bound antigen with the second antibody. Unbound second antibody is washed away, an enzyme substrate is added and the enzyme reaction is monitored. The amount of antigen in the test sample determined from the amount of bound second antibody.

Although the use of a solid substrate facilitates the separation of reactants in enzyme linked immunoassay techniques, a number of disadvantages exist. Uniform adsorption of the binding agent to the solid support is not easy to achieve. There is often non-specific adsorption of the antigen and/or enzyme labeled antibody to the solid-support during incubation which can lead to erroneous results. In addition, the sensitivity of these techniques is not the same as achieved by solution phase assays.

Radioimmunoassays, originally described by Yalow and Berson (1960, J. Clin . Invest . 39:157), are solution phase assays and are characterized by competing fixed amounts of radiolabeled analytes with unknown quantities of unlabeled analytes for fixed amounts of specific antibody. The amount of radioactive analyte either bound to antibody or free in solution is quantitated in an appropriate radioactivity counter and the concentration of non-radioactive analyte determined. Substrate- labeled fluorescent immunoassay involves covalent coupling of the analyte to a fluorogenic substrate for an enzyme. This analyte-substrate conjugate is not fluorescent. In the absence of antibody the analyte-fluorogenic substrate is hydrolyzed by an enzyme yielding a fluorescent molecular species. In the presence of specific antibody, access to the substrate by the enzyme is curtailed yielding decreased fluorescence (see U.S. Patent No. 4,708,929).

While radioimmunoassays (RIA) and fluorescent immunoassays (FIA) are more sensitive than the substrate bound assay techniques, these solution-phase assays have their own drawbacks . Radioimmunoassay technique requires the handling of radioactive materials and detection usually involves expensive equipment. Similarly, detection of fluorescence requires expensive and cumbersome equipment.

For soluble bindable molecules (e.g. antigens), the conditions under which it reacts with its affinity molecule (e.g. antibody) is critical for binding. For example, insulin, when bound to a substrate does not react efficiently with its antibody, making its detection by ELISA difficult. Towards that end, the most commonly used method of quantitating insulin in test samples, currently, is RIA. Similarly, a higher level of detection of insulin antibodies is achieved when the antibodies and insulin are allowed to react in a solution phase. For example, Kuglin et al . (1990, Workshop report of the Fourth International Workshop on the Standardization of Insulin Autoantibody Measurement) reported that RIA results from the sera of Type I diabetic patients showed significantly higher specific signals than those obtained by ELISA. It was observed that both the mean signal and the mean variance were higher in RIA than in ELISA In another study by Greenbaum et al, (1992, J^". Clin . Endocrinol . and Metab . 74:1040-1044), RIA and ELISA methods to detect insulin antibodies were compared. Experiments using the RIA method reported a much higher percentage of sera to be positive for insulin autoantibodies than experiments using ELISA. Consequently, fluid phase essays have been concluded to be superior to solid phase assays for identifying disease associated with insulin antibody levels .

The difference in detection abilities of substrate bound assays like ELISAs and solution-phase assays like RIA is considered to be related to the availability of epitopes. For example insulin antibodies may recognize various epitopes on the insulin molecule. In a solution-phase essay, most of the epitopes would be available for binding by an antibody. However, in a substrate bound assay, some of these epitopes may be involved in binding to the substrate or may encounter stearic hindrance due to their proximity to the substrate binding site . Such masking of binding epitopes may result in a failure to bind and consequently detect that molecule.

Insulin Dependent Diabetes Mellitus

Diabetes is a chronic and complex metabolic disease influenced by various hereditary and environmental factors that result in the inability of the body to maintain and use carbohydrates, fats and proteins. The condition, characterized by high blood glucose levels, is caused by a deficiency in insulin production or an impairment of insulin utilization. Most cases of diabetes fall into two clinical categories: insulin- dependent diabetes mellitus (IDDM or Type I diabetes) and non-insulin dependent diabetes mellitus (NIDDM or Type II diabetes) .

It is generally accepted that IDDM is an autoimmune disease targeting β-cells of the islets of Langerhans in the pancreas. What initiates the autoimmune response against β-cells is still unclear, but several recent studies have advanced scientific understanding of the disease . Much research has focused on the autoimmune response in diabetes with specific regard to immune markers. The immunologically mediated β-cell destruction in IDDM appears to be a slow process, indicated by the early appearance of immune markers like antibodies against islet cells (ICA) , insulin (IAA) , and 65kD glutamic acid decarboxylase (GAD) .

Perhaps the single most important advance in the past two decades of diabetes research has been the realization that autoimmune destruction of β-cells takes months or years to reach completion. Unfortunately, the clinical diagnosis of diabetes is almost never made until the destructive process is nearly complete. By this stage, potential preventive therapies (e.g. administration of anti-oxidants) are ineffective and one has to resort to insulin injections. Early diagnosis of the onset of diabetes mellitus will allow intervention in the progress of the disease at an early stage. Currently used methods for early detection of IDDM include testing for the presence of ICA. Unfortunately, these assays are flawed in many ways . They are cumbersome, unstandardized and difficult to adapt to mass screening. Moreover, the predictive value of ICA in the general population is expected to be low. The biochemical assays routinely used to detect anti-islet autoimmunity are radioimmunoassays for antibodies to insulin and immunoprecipitation assays for GAD and ICA- 512 autoantibodies . Independently, the occurrence of these autoantibodies in prediabetics was shown to be about 80% for ICA-512, 81% for insulin and 62% for GAD It is frequently observed that many prediabetics express two or more of these autoantibodies, while some express only one . With such a random assortment of autoantibodies in the prediabetic sera, some investigators have attempted to theoretically increase the positive predictive value for detecting diabetes among screened first degree relatives (and more dramatically in screening for general population) by evaluating more than one immune marker (e.g. ICA, IAA, anti-GAD, anti-ICA-512) . However, it has been difficult to adapt the complicated, time consuming biochemical assays for screening of the general population on a large scale by the currently available methods.

There is an ongoing need for sensitive assays for the detection of bindable molecules like antigens or antibodies in test samples. For example, it would be desirable to have a simple and inexpensive method, useful in mass screening, for the detection of antibodies or antigens that can be used for early detection of diseases before the onset of symptoms. Such a technique would allow effective intervention therapies at the early stages of diseases such as diabetes .

Summary of the Invention

The present invention provides a sensitive and simple method for detection and quantitation of analytes based on their binding to molecules (termed as detector molecules or first affinity molecules) that have a high affinity for the analyte. The detector molecules and the test sample containing the analyte are allowed to react in a solution phase. Following binding, the detector molecules are immobilized by means of pre- attached target molecules that have been covalently linked to the detector molecule. The target molecule may be attached to the detector molecule directly or via a spacer. The spacer enables the detector molecule to bind to the analyte without any significant stearic hinderance from the target molecule. Following incubation of the target molecule-spacer-detector complex with the test sample, the mixture is exposed to a solid matrix precoated with capture molecules having a specific affinity for the target molecule. The presence of spacer between the target molecule and the detector molecule also reduces any stearic hinderance for binding of the target molecule to the capture molecule. Following the capture' of target-spacer-detector complexes (either with or without attached analyte molecules) on the solid matrix, the amount of analyte immobilized is measured by incubation with a labeled second affinity molecule that binds specifically to the analyte. The amount of analyte in the test sample is calculated from the amount of label immobilized.

The method of the present invention can be used for the detection and quantitation of antibodies involved in autoimmune diseases, such as Insulin Dependent Diabetes Mellitus. The present invention, therefore, provides a sensitive and simple method for early detection of, and prediction of risk of Insulin Dependent Diabetes Mellitus . Thus, an object of the present invention is to provide a method for the detection of specific antibodies. A target molecule is attached to the antigen directly or via a spacer. A test solution containing antibodies is then incubated with the antigen. The incubation mixture is then poured on to a solid matrix pre-coated with capture molecules having specific affinity for the target molecule. The presence of bound antibody is detected by binding with a detectable second antibody that has affinity for the test antibody.

Another object of the present invention is to provide a method for the detection of antigens. An antibody specific for the antigen is used as the detector molecule. A target molecule is attached to the detector molecule via a spacer. Following binding of antigen and antibody in a solution phase, the bound complexes are immobilized to a solid matrix precoated with capture molecules. The antigen in the test sample is quantitated by exposing the complex to a detectable second antibody to the antigen. Similarly, ligand molecules can be detected by using their specific receptors as the detector molecules .

Another object of the present invention is to provide a method for the detection and quantitation of receptors. The detector molecule is a molecule that has a specific affinity for the receptor and thus includes its ligand or a mimic thereof, and specific antibodies to the receptor. In a preferred embodiment, the detector molecule is a ligand. Following binding of receptor to the ligand-spacer-target complex, the mixture is exposed to a solid matrix coated with capture molecules. Bound ligand-receptors can be detected by using a labeled antibody to the receptor.

Another object of the present invention is to provide a method for the detection of at least two antibodies to antigens selected from the group consisting of insulin, GAD and ICA-512. At least one autoantibody is detected by solution-phase ELISA while the other may be detected by solution-phase or solid- phase ELISA. A target molecule is attached to the antigen directly or via a spacer. A test sample containing antibodies is then incubated with the antigen. The sample may be incubated simultaneously or separately with the antigens. The incubation mixture (s) is then poured on to a solid matrix pre-coated with capture molecules having specific affinity for the target molecule. The presence of bound antibody is detected by binding with a detectable affinity molecule (e.g. anti-IgG) that has affinity for the test antibody. In one embodiment of the invention, all the antibodies are detected by solution-phase ELISAs A test sample is incubated with a mixture of insulin, GAD and ICA-512 either separately or together. A target molecule is attached to the antigen (detector molecule) via a spacer. Following binding of antigen and antibody in a solution phase, the bound complexes are immobilized to a solid matrix precoated with capture molecules. The autoantibodies in the test sample are quantitated by exposing the complex to detectable second antibodies specific for the autoantibody. A yet another object of the present invention is to provide kits for the detection of analytes and for the detection of IDDM using the methods disclosed herein.

Detailed Description of the Invention Definitions

By the term "bindable molecules", "bindable pair" or "affinity molecules" is meant for the purposes of specification and claims, to refer to a pair of compounds, in which one of the molecules has an affinity for and specifically binds to, and is therefore termed as complimentary to, a particular area of the other molecule. The bindable molecules include, but are not limited to, antigen-antibody and ligand-receptor complexes . By the term "analyte" is meant for the purposes of specification and claims, the molecule to be detected. This includes specific antibodies, antigens, ligands, and receptors. The antibodies may belong to any class such as IgA, IgM, IgG, IgD and IgE . Within IgG class, the antibodies may belong to various subclasses like IgGl, IgG2a, IgG2b and IgG3.

By the term "antigen" is meant for the purpose of specification and claims, a compound against which antibodies can be raised, and which is capable of binding to an antibody to form specific antibody-antigen complexes. The antigen may be natural or synthetic and further may be of prokaryotic or eukaryotic origin. Synthetic antigens may include drugs, pesticides and the like. The antigen may be used in its natural form or may be modified so as not to affect its binding to the specific antibody. Typical modifications include covalent or non-covalent modifications to a detectable label or to an attachable molecule. A modified antigen also includes a molecule that is a fusion protein. Fusion proteins may be formed by the fusion of portions of two or more peptides from the same or different species .

By the term "ligand" is meant for the purposes of specification and claims, a compound for which a receptor naturally exists or can be prepared. The ligand may be natural or synthetic and further may be of prokaryotic or eukaryotic origin. Synthetic ligands may include drugs, pesticides, antibiotics and the like for which receptors exist. The ligand may be used in its natural form or may be modified so as not to affect its binding to the specific receptor. Typical modifications include covalent or non-covalent modifications to a detectable label or to an attachable molecule.

By the term "receptor" as used herein for the purpose of specification and claims is meant a compound, whether naturally occurring or not, that recognizes an epitope or a determinant site on, and is capable of binding to, the determinant site of a complimentary molecule termed its ligand. The receptors may be bound to other materials, i.e. protein receptors bound to membranes or the receptors may be soluble like steroid receptors .

By the term "spacer" is meant for the purpose of specification and claims, a molecule generally linear in three dimensional configuration that has binding capacity at both ends. Thus, a spacer can bind to a detector molecule on one end and a target molecule on the other with the result that the detector molecule as well as the target molecule are free from the stearic hindrance that would be created by the binding of the detector molecule directly to the target molecule. The spacer may be bound to the target molecule either before or after binding of the detector molecule to one end. Various spacers are well known in the art. A molecule comprising a chain of carbon atoms is an example of a suitable spacer. The length of the spacer is not limited to any particular number of carbon atoms.

By the term "target molecule" is meant for the purposes of specification and claims, a member of a complimentary pair that will allow the immobilization of the detector molecule to a solid matrix. Target and capture complimentary pairs may be immunological pairs like antigen-antibody e.g. biotin-antibiotin, horseradish peroxidase (HRP) -antiHRP, fluoroisothiocynate (FITC) -antiFITC, or non immunological pairs like biotin-avidin, biotin- strepavidin, and magnetic beads-iron traps.

By the term "capture molecule" is meant for the purpose of specifications and claims, a molecule that exhibits specific affinity for the target molecule. Thus, the capture molecule may be a specific antibody, a non-immunological molecule (e.g. avidin or strepavidin), or a magnetic substance.

By the term "detector molecule" or "first affinity molecule" is meant for the purposes of specifications and claims, a molecule having a high affinity for the analyte. Thus, a detector molecule is one of the members of a bindable pair. For example if the analyte is an antibody, the detector molecule may be its antigen. The detector molecule may be natural or synthetic and includes completely folded globular proteins, e.g. secreted polypeptides, receptors, antibodies, unfolded fragments or peptides of any length of proteins including globular proteins; organic molecules that are composed of and are derivatives of the 5 or 6 carbon atom ring compounds; antigens of any disease causing organism including double and single stranded nucleic acids; and all sugar and carbohydrate moieties. When an antibody is used as a detector or an affinity molecule (both a first and second affinity molecules) , the antibody may be a complete molecule or it may be a fragment of the antibody like Fab, F(ab)2 and Fv fragments. The detector molecule may be a complete molecule or a fragment thereof containing the epitope recognized by the analyte.

By the term "antibody fragment" or "fragment thereof" referring to an antibody, as used herein, is meant for the purposes of the specification and claims, to mean a portion of fragment of an intact antibody molecule, wherein the fragment retains antigen-binding function; i.e. F(ab')₂# Fab', Fab, Fv, single chain Fv ("scFv), Fd' and Fd fragments. Methods of producing the various fragments from antibodies are well known in the art .

The present invention relates to a high sensitivity method and kits for detection and quantitation of analytes in which the high sensitivity of liquid immunoassays is combined with the ease and cost of solid phase assays. An analyte is detected via its binding to a 'detector' molecule which has a high affinity for the analyte. The high sensitivity is achieved by allowing the analyte and detector molecules to react with each other in solution. Subsequent to its binding with the analyte, the detector is immobilized. This is in contrast to conventional techniques wherein the analyte is allowed to react with an immobilized detector molecule. In the present invention, to facilitate immobilization of the detector molecule following binding with the analyte, a second pair of affinity molecules comprising of a target molecule and a capture molecule is utilized. The target molecule and the capture molecule have specific affinity for each other. One member of this pair, the target molecule is preattached to the detector molecule while the other member of the pair, the capture molecule is attached to a solid substrate. Thus, after allowing the affinity molecules (analyte and the detector-target complex) to interact in a solution phase, bound complexes are immobilized via attachment of the target molecule to a solid matrix that is precoated with the capture molecule. It is preferable to introduce a spacer between the detector and the target molecule so that the target molecule does not pose a stearic hindrance for binding of the detector molecule (or vice versa) to the analyte. The immobilized complexes containing analyte molecules are then detected and quantitated by using a labeled second affinity molecule that has a specific affinity for the analyte. Preferably, the second affinity molecule binds to a different epitope of the analyte than the first affinity molecule i.e. the detector molecule. Thus, bound complexes comprising capture molecule-target molecule-detector molecule can be distinguished from bound complexes comprising capture molecule-target molecule-detector molecule-analyte . An important consideration in the assay of the present invention is the specificity of binding of the analyte to the selected detector molecule . The detector molecule should have a high affinity for the analyte.

It can be any organic or inorganic compound that has an affinity for the analyte. Generally, the detector molecule is a peptide or a polypeptide To achieve a high degree of specificity, the affinity molecule should be in a substantially pure form. Affinity molecules in a purified form, may be purchased commercially or purification can be accomplished by standard techniques well known in the art of protein purification including detergent extraction, chromatography (e.g. ion exchange, affinity, immunoaffinity, or sizing columns) , differential centrifugation, and differential solubility. Immunopurification of polypeptides may be accomplished using methods known in the art for immunoaffinity chromatography. Monoclonal antibodies specific for epitopes of that polypeptide may be linked to a chromatographic matrix to form an affinity matrix. The preparation containing the polypeptide is then incubated with the affinity matrix allowing the antibodies to bind to the polypeptide. Unbound components are removed by extensive washing of the matrix and the polypeptide is eluted from the matrix. The polypeptides can be used as such or can be cleaved to smaller peptides using methods known in the art . In addition, fusion proteins may also be used as detectors molecules for antigenic epitopes that are too small to act as efficient detector molecules. Further, fusion proteins (for example with glutathione-S-transferase) produced by various expression systems may also be used. A key feature of this invention is to provide means of easy separation of bindable affinity molecules after allowing the molecules to react in solution. To be useful for the present invention, target molecule should not only have an affinity for the capture molecule but should also be covalently attachable to the detector molecules. Examples of suitable target molecule - capture molecule pairs include both immunological pairs (e.g. antigen-antibody) and nonimmmunological pairs (e.g. biotin-avidin) .

In a preferred embodiment, to prevent stearic hindrance from the target molecule to the binding of the detector molecule with the analyte, a spacer is used to separate the target molecule from the detector molecule. The spacer also reduces any stearic hindrance from the detector-analyte complex to the binding of target molecules to the capture molecules . A spacer is typically a bifunctional molecule that contains two reactive sites. Various spacers differing in types of reactive groups, hydrophobicity or hydrophilicity, and length of the structure connecting the reactive groups have been described in the literature (see Meth. Enz . , 1983, 91:580-609), and are well known to those skilled in the art . Suitable spacers have been described in U.S. patent number 5,667,764, which is hereby incorporated by reference, and include straight and branched chain carbon spacers, heterocyclic carbon spacers, and peptides. The functional linkage of the spacer may be any reactive group including an amide (- NHCO) , thriourea (-NHCSNH-), hydrazone (=NHN-), acyl hydrazone (=NHNC0) , ketal ( -0-C (alkyl) 2-0- ) , acetal (-0- CH (alkyl) -0-) , orthoester ( -C (0-alkyl) 2-0- ) , ester (- C00-) , anhydride (-C00C0-), disulfide (-S-S-), urea (- NHC0NH-) , carbamate ( -NH-CC=0) -0- ) , imine (=N-), amine (-NH-) , ether (-0-), carbonate ( -0-C (=0) -0- ) , thioester (-S-), sulfonamide (-S0₂-NH-), carbonyl (-C0-) and amidine ( -NHC= (NH (+)-)) .

An important consideration in the selection of a spacer is chain length. The importance of spacer chain length for retention of activity of binding partners has been demonstrated in enzyme-substrate reactions. A six- carbon spacer exhibited an activity of 12% of that of soluble enzyme while a two-carbon spacer exhibited an activity of 3.2% of that of the soluble enzyme (Kennedy and Cabral, 1987, Meth . In Enzymology, 135:117-130). In another study, a glutaric anhydride cross-linker produced 30% of the soluble enzyme activity for papain versus a 5% activity for directly coupled papain. Maximum activity retention was observed when papain was cross-linked via polyethylene glycol (PEG 600) cross- linker (Jayakumari and Pillai, 1991, J^". Appl . Polym .

Sci . , 42:583) . For the present invention, the spacer may be straight or branched one. It is preferable to use a straight chain spacer of 3-18 carbons However, optimal chain length will vary depending upon the nature of the protein or peptide involved. The determination of optimal chain length for a particular peptide can be obtained by standard binding techniques known to those skilled in the art. Spacers can be attached to the molecule of interest by commercially available kits.

In the present invention, it is contemplated that one end of the spacer is bound to the detector molecule which may be an antigen, antibody, ligand, or a receptor. The other end of the spacer is attached to a target molecule by which the detector can be immobilized. It is important that the binding of the spacer itself to the detector be achieved at the site other than the one involved in the subsequent binding to the analyte. Stearic hindrance from the spacer itself is unlikely unless the spacer is attached at, or extremely close to, the sight of analyte binding.

For the binding of the capture molecules to a solid matrix, it is preferable to achieve a uniform adsorption of the capture molecules to the solid substrate. Thus, the conditions during the process of attachment to the substrate are kept constant so as not to introduce changes in the ionic charge of the substrate environment. Various solid matrices are known in the art for binding techniques. Suitable solid matrices include but are not limited to, plastic or polyvinyl microtiter or culture wells, polystyrene or magnetic beads, membranes like nitrocellulose or poly vinylidene di- fluoride, or columns that are packed with various matrices.

For the detection and quantitation of analytes according to the method of the present invention, the detector-spacer-target ( ' dst ' complex) complex is incubated with the test sample. The sample to be analyzed includes any liquid sample. Thus, for example, it may be a body fluid including blood, serum, plasma, saliva, cerebrospinal fluid, urine and the like, or may be a tissue culture sample. The sample may be used as such or may be partially purified. Binding is carried out in standard binding buffers. Such buffers are well known to those skilled in the art. Some suitable buffers include phosphate, tris, glycine, citric acid, and sodium acetate. The molarity of buffers used in binding assays generally ranges from 0.001 to 0.3 M. Various blocking or coating molecules, or surfactants may be added to the binding solutions to reduce nonspecific binding. Suitable blocking agents include proteins like albumins, gelatin, nonspecific IgG, nonfat dry milk, and surfactants like Tween 20, Tween 80, and Triton X-100. For each analyte, optimal binding conditions can be determined by techniques well to those skilled in the art. These include variation of buffer, pH, ionic conditions etc. Following binding of detector molecules to any analyte molecules in the sample, the reaction mixture is poured onto a solid substrate pre-coated with capture molecules. The analyte-dst complex will be 'captured' on the solid substrate. It is preferable to include some blocking agents in the incubation mixture to reduce nonspecific binding. Following incubation of the reaction mixture with solid matrix, unbound materials are removed by extensive washing with a buffer. It is preferable to include some blocking agents in washing solutions. It should be noted that capture molecules will 'capture' dst complexes with or without attached analyte molecules. Bound analytes are then detected by using a reporter system which includes a second molecule that has a specific affinity for the analyte. For example, when the analyte is of human origin, an anti-human IgG having a detectable label may be used. The detectable label may be an enzyme including, but not limited to, alkaline phosphatase, β- lactamase, β-galactosidase, urease or horseradish peroxidase; a fluorochrome, a radionucleotide, or a latex or gold particle. Preferred enzymes are alkaline phosphatase and horseradish peroxidase. Various enzyme substrates or chromogens are known in the art including p-nitrophenyl-phosphate, 5-bromo-4-chloro-3 - indolyl- phosphate, 3 , 3-diaminobenzidine, and o-phenylenediamine .

After removal of the unbound labeled antibody by washing with a buffer, the quantity of bound label remaining on the solid matrix is directly related to the amount of analyte originally present in the test fluid. Accurate quantitation may be achieved by using various dilutions of the test sample and determination of the concentration from a standard curve. Such quantitations are well known to those skilled in the art . The color intensity of the solid matrix may be compared visually to a color guide for a qualitative or semi-quantitative detection or measured quantitatively using absorbance/reflectance photometry well known in the art. For example, when the solid matrix is a microtitre plate, a commercially available plate reader can be used for generating quantitative data.

In one embodiment of the invention, the first affinity molecule for the analyte i.e. the detector molecule as well as the second affinity molecule for the analyte i.e. of the reporter system, are allowed to react in a solution phase. Thus, a test sample is incubated with the detector-spacer-target complex. The second affinity molecule may be added at the same time or after allowing the dst complex-analyte reaction to reach equilibrium. Following appropriate incubation, the mixture is poured on to a solid matrix pre-coated with the capture molecules. After incubation, unbound materials are washed off and the amount of label bound is detected. In another embodiment of the invention, provided is a kit for a method for the detection of an analyte, wherein the kit comprises a first affinity molecules having a specific affinity for the analyte; a spacer; a target molecule; a capture molecule; and a reporter system comprising a second affinity molecule having a specific affinity for the analyte and having a detectable label.

In an additional embodiment of the invention, provided is a kit for a method for the detection of an antibody, wherein the kit comprises a detector molecule (insulin, GAD and ICA-512) having a specific affinity for the antibody; a spacer; a target molecule; a capture molecule; and a labeled affinity molecule having a specific affinity for the antibody. In another embodiment of the kit are included a monoclonal antibodies for 6-His ICA-512, and 6-His ICA-512.

The following examples are presented to further illustrate the various embodiments but are not meant as a limitation.

Example 1

Formation of tarcret-spacer-detector complex

Spacers can be covalently attached to a detector molecule on one end and a target molecule on the other end by techniques well known to those skilled in the art .

In one illustration of this embodiment, biotin was used as a target molecule. Biotin labeling of detector molecules can be carried out by using N- hydroxysulfosuccinimide ester chemistry to attach biotin to a primary amine on the protein. Biotinylation of proteins is carried out by procedures well known in the art. For example, 0.1 to 1.0 mg/ml solution of protein or peptide in borate buffer (pH 8.0-8.5) is incubated with a freshly prepared solution of biotin in DMSO . After incubation at room temperature, free biotin is removed by extensive dialysis against a buffer such as phosphate buffered saline. In another illustration of this embodiment, a water-soluble derivative or biotin e.g. N-hydroxysuccinimide ester, can be used.

Biotinylation kits are available commercially (Pierce) and proteins can be labeled with biotin by following manufacturer's instructions. Biotinylated detector molecules can be separated from free biotin using molecular exclusion chromatography column e.g. G-50. In addition, some biotinylated proteins are available commercially.

In another illustration of this embodiment, a water soluble derivative of biotin that has a preattached caproyl arm (NHS-LC-biotin from Pierce) was attached to insulin. Insulin solution was prepared and biotinylated according to manufacturer's instructions.

Example 2 Attachment of Capture Molecules to Solid Matrices

The attachment of capture molecules to a solid matrix can be achieved by non-covalent or covalent means. Many different protocols are known in the art to attach molecules to a solid surface. For example, capture molecules in solution are added to the solid substrate . To prepare the solid surface for subsequent addition of target labeled detector molecule/analyte complex, the solid surface must be washed to remove unbound capture molecules from the coating surface. Non-specific sites, defined as uncoated surface on the solid matrix which could bind nonspecifically to components added subsequently, are saturated or blocked to reduce background. Blocking agents, for reducing nonspecific binding, are well known in the art and include proteins like bovine serum albumin, gelatin, and detergents like tween-20 and triton X-100, and combinations thereof. For blocking, a solution containing the blocking agent is added to the solid substrate following coating with the capture molecule. Excess blocking agent is removed by washing the solid surface with a buffered solution.

In one illustration of this embodiment, avidin was coated on to microtiter plates. The coating was achieved by passive adsorption in a suitable buffer. For coating, 1.6 μg of avidin in a buffer (phosphate buffered saline, Sigma) was added to each well of a 96 well microtiter cluster (Nunc Inc, Naperville, IL) in glycine buffered saline (pH 8.6) at 4°C overnight. Unbound avidin was removed by washing the plates three times in the glycine buffered saline with an automatic plate washer (Model Well wash 4, Denley Instruments,

Morrissville, NC) . Avidin coated plates can be stored in sterile PBS for up to one year and used when needed.

Example 3 Comparison of solution phase ELISA to conventional solid phase ELISA

This embodiment demonstrates that solution phase ELISA of the present invention is more sensitive than the conventional solid phase ELISA. To illustrate this embodiment, detection of insulin antibodies in human serum samples was carried out . Although any molecule that has an affinity for the analyte to be detected can be used as a detector molecule, in this illustration of the embodiment, insulin was used as the detector molecule. No difference in the quantitation of insulin antibodies was observed between human insulin and bovine insulin, and therefore, bovine insulin was used in subsequent experiments because of its relatively low cost. Insulin was biotinylated via a caproyl arm as described in Example 1. Biotinylated insulin (50ng to 350ng per reaction mixture) was incubated with a 1:100 dilution of serum in a buffer diluent. It is preferable to is run each sample in duplicate or triplicate. After incubation at 37°C for one hour, the incubation serum mixture was added to microtiter plates precoated with avidin as described in Example 2. After incubation at 37° for 90 minutes, wells were washed four times with PBS. Goat anti-human IgG and IgM antibodies (Jackson Laboratories) conjugated to alkaline phosphatase was added to the wells at a dilution of 1:400 and incubated at room temperature for 30 minutes. The wells were washed four times with PBS. A substrate of alkaline phosphatase (p-nitrophenyl phosphate, pNPP) was added to the wells at a concentration of 0.1% pNPP . After 20 minute incubation at room temperature, the reaction was stopped with 5% Ethylene diamine tetra acetic acid

(EDTA) , tetrasodium salt. The absorbance of the wells was measured at wavelengths of 405/630 nm. The optical density of samples for solid phase ELISA and solution phase ELISA is shown in Table 1. Samples above a Mean ± 3 standard deviation cutoff are considered positive. The cutoff for the solid phase assay was 0.365 and for the solution phase assay was 0.352. As shown in Table 1, while only 9 samples out of 20 tested were deemed 'positive' by the conventional solid-phase ELISA, 18 out of the 20 samples tested were deemed 'positive' by the solution phase ELISA for insulin antibodies. Thus, this embodiment, demonstrates that solution-phase ELISA of the present invention is more sensitive than the conventional solid phase ELISA. Table 1

Example 4

Use of target -capture molecule improves the sensitivity of solution phase ELISA This embodiment illustrates the unexpected finding that the solution phase ELISA of the present invention is more sensitive when the detector-spacer complex is immobilized to a solid substrate via a target molecule - capture molecule pair than when it is immobilized directly. To compare ELISA results with and without the target-capture molecule pair, insulin was biotin labeled via a caproyl arm and then attached to a microtiter plate via avidin or strepavidin coating (Figure 2a) , or attached directly to the microtiter plates using the N- hydroxysuccinimide chemistry (Cova Link plates, Nunc Inc, Naperville, IL) , Figure 2b. Solution phase ELISA was carried out as described in Example 3. Table 2 presents results of the experiments to detect insulin antibodies. The results are expressed as optical density (O.D.) for the corresponding sera. The mean value for 64 normal human sera samples using COVA- link was 0.382 and using avidin was 0.279. Samples above the mean value were considered to be positive for anti- insulin antibodies. As seen in Table 2, binding of insulin-antibody complex via a spacer and avidin-biotin revealed many more samples to be positive than binding via the spacer alone . This indicates that the use of capture-target molecule pair increases the sensitivity of the solution phase ELISA assay.

In another illustration of this embodiment, experiments were conducted to determine if there was any difference between using two different capture molecules known to have affinity for biotin. One of the molecules selected was avidin. Another molecule, strepavidin, is known to bind avidin with greater affinity than avidin. Solution phase ELISA was carried out as described in Example 3 to quantitate insulin antibodies in human sera. No difference was observed between the results obtained with avidin and strepavidin (data not shown) . Table 2

Example 5

Detection of antigens

This embodiment illustrates the detection of antigens using solution phase ELISA of the present invention. For the detection of an antigenic molecule, a specific antibody or a fragment thereof to the antigen is used as a detector molecule and is attached to a target molecule like biotin via a carbon spacer. The biotinylated antibody is then allowed to react with the test solution. Following adequate incubation to achieve binding, the mixture is added to a solid matrix like a microtitre plate that has been coated with a capture molecule (like avidin or strepavidin) . The antigen- antibody complex is immobilized to the solid matrix. A second antibody or another affinity molecule (a receptor molecule) or a fragment thereof having a specific affinity for the antigen and that has an enzyme label on it is then added to the microtitre plate. After removing the unbound materials, the amount of antigen in original sample can be determined from the amount of enzyme label bound. It is preferable to use monoclonal antibodies as both the first and the second antibodies. In addition, it is important that the second antibody is directed towards an epitope different than the first one so that the binding of one antibody does not interfere with the binding of the other.

In one illustration of this embodiment, serum insulin levels can be quantitated. Briefly, an antibody to insulin is attached to a target molecule (biotin) via a spacer. A plasma or serum sample is incubated with the biotinylated anti- insulin antibody. Preferably, incubation is carried out at between room temperature to 37°C for 10-60 minutes. The mixture is added to strepavidin coated plates and further incubated (room temperature for 10-60 minutes) . Unbound materials are washed out and a labeled second antibody directed to a different epitope of insulin than the first antibody is added to the plates. A standard graph is generated by using a range of known concentration of insulin. The amount of insulin in the test sample can be computed from the standard graph.

In the detection of antigens, it is preferable to obtain antibodies in a pure form. Both monoclonal and polyclonal antibodies can be used for detection of the antigen. Monoclonal antibodies offer several advantages over polyclonal antibodies. For example, mABs, using the techniques pioneered by Kohler and Milstein, 1975, Na ture, 256:495-97) can be obtained in large quantities and in highly pure form. In storage, their activity is retained over time. Hybridoma cells, which produce monoclonal antibodies, can be easily stored over a long period of time without losing their ability to produce the mABs. In addition, mABs do not exhibit an ongoing need for the antigen or to obtain blood from the immunized animal .

Example 6

Detection of Antibodies For the detection of antibodies in the test sample, an antigen that the antibody is directed towards is attached to a target molecule via a spacer. The test sample in incubated with the antigen-spacer-target complex. The reaction mixture is exposed to a solid substrate precoated with capture molecules. After removing unbound antibody, a labeled second antibody directed towards the test antibody (e.g. anti-human IgG, if the test sample is human) is added. The second antibody may be the whole molecule or may be fragments of the antibody.

The solution phase ELISA can be adapted to detect various classes of antibodies. Thus, in situations where quantitation of IgG and IgM may be required (as in detection of diabetes) , the assay can be adapted wherein the reporter system comprises labeled polyclonal antibodies that recognize both the IgG and IgM molecules or a mixture of monoclonal antibodies that specifically recognize IgG or IgM may be used. Detection of Antibodies Related to IDDM In one illustration of this embodiment, antibodies to insulin in human serum samples were detected and quantitated using the solution phase ELISA of the present invention. The methods and results of these experiments are discussed in Example 2. In another illustration of this embodiment, antibodies to GAD in human serum samples were quantitated using solution phase ELISA of the present invention. The GAD protein can be commercially purchased in a pure form (Synectics Biotechnologies, Sweden) or can be purified from recombinant clones using techniques well known in the art. To further illustrate this embodiment, GAD protein was purchased from a commercial source and biotinylated using a commercially available kit (Pierce) following the manufacturer's instructions. Briefly, N-hydroxy Succinimide biotin was incubated with GAD protein solution (0.6mg/ml) for 30 minutes at room temperature. The reactants were intermittently vortexed gently during incubation. The mixture was dialyzed using molecular exclusion chromatography (PD10 columns, BIORAD) . Fractions were collected and assayed for protein content using a commercially available protein estimation kit (Pierce BCA protein) . Fractions containing the GAD protein were tested for biotinylation as follows . A small aliquot of each fraction containing approximately lOug/ml of GAD protein was added to duplicate wells precoated with 16 μg/ml avidin. The wells were incubated for 30 minutes at room temperature, washed several times with a buffer (PBS) and incubated with 100 μl/well strepavidin- alkaline phosphatase (Jackson Labs, 1:500 dilution) for 30 minutes at room temperature. Following washing of the wells, pNPP substrate was added (lOOul/well) , incubated for 10 minutes at room temperature and the reaction was then stopped by adding 5% EDTA. Fractions containing biotinylated GAD were used for anti -GAD solution phase ELISA according to the method of the present invention. Biotinylated GAD (125ng) in solution was incubated with 1:100 dilution of sera for 1 hour at 37°C. The mixture was transferred onto avidin coated microtiter plates and incubated for 90 minutes at room temperature. The plates were washed and bivalent IgG and IgM alkaline phosphatase conjugate was added. Color development upon addition of substrate indicate the presence of antibodies to GAD.

In another embodiment of this invention, antibodies to the antigen ICA-512 are detected using the solution phase ELISA of the present invention. For detection of ICA 512 antibodies, ICA-512 can be purified by methods known in the art. For example the method of Rabin (U.S. Patent 5,200,318), which method is hereby incorporated by reference can be used to clone and purify ICA-512. In one illustration of the embodiment, the protein was purified by expressing the plasmid containing the full length sequence of the protein in a bacterial strain. To increase sensitivity, it is preferable to express clones containing the full length rather than truncated versions of the ICA-512.

In another illustration of the embodiment, a plasmid is constructed in such a way that the expressed protein has a six-histidine tail at the C-terminal end of the ICA-512 molecule. ICA-512 protein containing a 6-His tail can be expressed and purified by standard techniques known to those skilled in the art. For example, plasmid pRSET-C was transduced in a bacterial strain (HMS174 (DE3 ) pLys of Escherichia col i ) . Colonies were plated, and positive clones selected and expanded on suitable medium (LB/Ampicillin medium) . Recombinant ICA-512 protein was expressed by stimulation with 1 mM IPTC in super broth containing ampicillin. Protein was extracted from cell pellets in a lysis buffer containing detergents (for example Triton X-100, PMSF, AEBSF, Leupeptin, and DNase) and purified by affinity columns having affinity for 6 His portion of the recombinant proteins (Talon-metal columns) .

Purified ICA-512 is biotinylated as described in Example 1. Briefly, ICA-512 is biotinylated using a commercially available kit (Pierce) following the manufacturer's instructions. Briefly, N-hydroxy Succinimide biotin is incubated with ICA-512 protein solution (0.6mg/ml) for 30 minutes at room temperature. The reactants are intermittently vortexed gently during incubation. The mixture is dialyzed using molecular exclusion chromatography (PD10 columns, BIORAD) .

Fractions can be collected and assayed for protein content using a commercially available protein estimation kit (Pierce BCA protein) . Fractions containing the ICA-512 protein are tested for biotinylation as follows. A small aliquot of each fraction containing approximately lOug/ml of GAD protein is added to duplicate wells precoated with 16 μg/ml avidin. The wells are incubated for 30 minutes at room temperature, washed several times with a buffer (PBS) and incubated with 100 μl/well strepavidin-alkaline phosphatase (Jackson Labs, 1:500 dilution) for 30 minutes at room temperature. Following washing of the wells, pNPP substrate is added (lOOul/well) , incubated for 10 minutes at room temperature and the reaction is then stopped by adding 5% EDTA. Fractions containing biotinylated ICA-512 were used for anti-ICA-512 solution phase ELISA according to the method of the present invention. Biotinylated ICA-512 (125ng) in solution was incubated with 1:100 dilution of sera for 1 hour at 37°C. The mixture was transferred onto avidin coated microtiter plates and incubated for 90 minutes at room temperature. The plates were washed and bivalent IgG and IgM alkaline phosphatase conjugate was added. Color development upon addition of substrate indicate the presence of antibodies to ICA-512.

Alternatively, in another illustration of the embodiment, ICA-512 antibodies can also be detected by solid ELISA in which ICA-512 is captured onto a solid matrix by an affinity molecule that is precoated on to the matrix. The affinity molecule may be a monoclonal antibody to ICA-512 or may be an antibody to an extraneous tail attached to one end of ICA-512. For example, a monoclonal antibody to 6-His can be used to capture 6-His ICA-512 antigen on the ELISA plates. The presence of ICA-512 autoantibodies can be detected in test serum by adding it to the solid matrix. Bound materials are detected by using an antibody to ICA-512 and labeled antihuman antibodies.

In a further illustration of the embodiment, 50 ng of a anti-6-His murine monoclonal antibody in carbonate buffer (pH 9.6) was used to coat a standard microtitre plate. Purified ICA-512 was added to the microtitre plates at a dilution of 1:100. After removal of unattached protein, test sample was added to the plate for 1 hour. Unbound materials were removed by washing and bound complexes were detected by using anti-ICA-512 and a second labeled antibody to anti-ICA-512. Combination ELISA

For the detection of IDDM, each of the three ELISAs (for insulin, GAD and ICA-512) can be used individually or in combination wherein the presence of autoantibodies for more than one antigen is detected simultaneously in the same assay. For example, to detect autoantibodies to all three antigens simultaneously, test samples (for example, serum samples) are incubated with biotin labeled insulin, GAD and ICA-512 either separately or together. The incubation mixture is then poured onto an avidin or strepavidin coated plate. Following incubation and removal of unbound materials, bound complexes containing autoantibodies can be detected using a mixture of labeled antibodies directed to the autoantibodies. For example, a labeled anti -IgG would detect bound autoantibodies (IgG) to insulin, GAD and ICA-512.

The combination assay may also comprise of a solution phase ELISA for one or two autoantibody and a Solid phase or conventional ELISA for the other (s) . To further illustrate this embodiment, a combination ELISA comprising solution phase ELISAs for insulin and GAD autoantibodies and a solid phase ELISA was used for ICA- 512. A solid matrix (microtitre plate) was coated with anti- 6 His monoclonal antibody and avidin either simultaneously or sequentially. Unbound materials are removed by washing and unreacted sites are blocked to reduce nonspecific binding. Test serum is incubated with biotinylated insulin (from Example 2.) and biotinylated GAD (from Example 2) either separately or in the same sample and added to each solid matrix. Unbound materials are removed by washing and labeled second affinity molecule (goat anti-human IgG and IgM conjugates are added. In an illustration of the sequential coating of 6- His monoclonal antibody (mAb) , and avidin, microtitre plates were coated with 6-His mAb (50 ng) overnight at 4°C. Unbound materials were removed by washing with PBS. The microtitre plates were then coated with 16 ug/ml avidin overnight at 4°C. For simultaneous coating, both the avidin solution and the 6-His ICA-512 solution can be added to the wells at the same time. Unbound avidin was removed by washing with PBS. ICA-512 was added to each well (1 ug/well) . Unbound ICA-512 was removed by washing with PBS. To reduce nonspecific binding, the microtitre plates were incubated with 1% calf serum (300 ul/well) for one hour at room temperature and washed with PBS. A 1:100 dilution of the serum sample was incubated with insulin-biotin and GAD-biotin as described in Example 3. Following incubation, the sample was added to the microtitre plates and further incubated. Unbound materials were removed by washing with PBS and bound materials are detected by the addition of a labeled second antibody (e.g. anti human IgG and IgM conjugated to alkaline phosphatase) . As shown in table 3, combination ELISA is able to detect individuals who are positive for only one marker but negative for others. For example, samples 4,9, 10,13,16,18 and 21 are negative for some antibodies but positive on the combination ELISA of the present invention.

Table 3

* indicates positive results Detection of Other Antibodies

In yet another illustration of this embodiment, antibodies to the pyruvate dehydrogenase complex were detected in serum using the solution phase ELISA of the present invention. The PDH-E2 gene was cloned using the pGEX-2T vector system. The PDH-E2/Glutathione-S Transferase fusion protein was used for detecting PDH autoantibodies .

In the assay for autoantibodies to the PDH complex, the presence of antibodies specifically directed to the E2 subunit was determined as follows. The PDH-E2/GST complex was biotinylated via a spacer using the biotinylation kit from Pierce. Biotinylated PDH-E2 (60ng) in solution was incubated with 1:100 dilution of sera for 1 hour at 37°C. The mixture was transferred onto avidin coated microtiter plates (coated with 16 μg/ml) and incubated for 90 minutes at room temperature. The plates were washed and 1:400 dilution of IgG alkaline phosphatase conjugate was added. Color development upon addition of substrate indicated the presence of antibodies to PDH-E2. The results of detection of antibodies to PDH-E2 are shown in Table 4a and Table 4b. Table 4a shows the use of conventional ELISA and comparison to anti-mitochondrial antibodies (AMA's) detected by using a commercially available immunofluorescence kit ( IMMCO Diagnostics) . In a total sample comprising 91 samples, 71 were negative by both immunofluorescence (IF) and standard ELISA. One sample that was positive by IF, did not show a positive reaction in the standard ELISA. Further, there were 7 false positives as seen by a negative scoring by IF but a positive reaction on the ELISA. Thus the standard ELISA not only fails to detect some positive samples but also gives erroneous results in false positives. A similar experiment with a different set of samples when analyzed by solution-phase ELISA of the present invention (Table 4b) showed no false negatives i.e. all samples that tested positive by IF also tested positive by the solution phase ELISA. Further, the number of false positives (positive on solution phase ELISA test but negative on the IF) were significantly reduced. These results indicate that the solution phase ELISA of the present invention is more sensitive and specific than the conventional solid phase ELISA.

Table 4a

Table 4b

Example 7

Detection of Receptors This embodiment illustrates quantitation of receptors or receptor-like molecules by solution phase ELISA of the present invention. The receptors that can be quantitated by the method of the present invention include, but are not limited to, Fc receptors of all classes, receptors for neurotransmitters (e.g. dopamine, muscarinic and acetylcholine) , hormone receptors (for both peptides and steroids) . Also included in this category of receptor- like molecules are ion channel proteins imbedded in cell membranes. For the detection of receptor molecules in a test sample, a ligand for the receptor is used as the detector molecule. The ligand can be attached to a target molecule via a spacer. The test sample is then incubated with the ligand-spacer- target molecule complex. The incubation mixture is added to the solid substrate precoated with capture molecules. After removal of unbound materials, an enzyme- labeled antibody directed towards the receptor is added. Preferably, the enzyme-labeled antibody is directed towards an epitope distinct from that involved in binding to the ligand, and the binding of the receptor to the ligand does not stearically hinder binding of the antibody to the receptor. The antibody may be a complete molecule or may be a fragment of the whole antibody.

From the foregoing, it will be obvious to those skilled in the art that various modifications in the above-described methods and compositions can be made without departing from the spirit and scope of the invention. Accordingly, the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Present embodiments and examples, therefore, are to be considered in all respects as illustrative and not restrictive, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

What is claimed is:

1. A method for quantitation of an analyte in a test sample comprising the steps of a) contacting the analyte with a first affinity molecule to form a bound complex, wherein the first affinity molecule has a specific affinity for the analyte and is covalently linked to a target molecule via a carbon spacer; b) immobilizing the bound complex to a solid matrix, said solid matrix being precoated with a capture molecule, wherein the capture molecule has a specific affinity for the target molecule; c) contacting the immobilized complexes with a labeled second affinity molecule to label immobilized complexes containing the analyte, wherein the second affinity molecule specifically binds to the analyte; d) removing unbound labeled second affinity molecule; and e) quantitating the amount of analyte in the sample from the amount of label immobilized.

2. The method of claim 1, wherein the carbon spacer is selected from the group consisting of straight chain carbon spacer, branched chain carbon spacer, and heterocyclic carbon spacer.

3. The method of claim 2, wherein the spacer is a straight chain carbon spacer and has a chain length of 3 to 20 carbon atoms.

4. The method of claim 3, wherein the spacer has a chain length of 13 carbon atoms.

5. The method of claim 1, wherein the label on the second affinity molecule is selected from the group consisting of enzyme label, radioactive label, and fluorescent label .

6. The method of claim 5, wherein the enzyme label is selected from the group consisting of alkaline phosphatase, horse radish peroxidase, ╬▓-lactamase, ╬▓- galactosidase, and urease .

7. The method of claim 1, wherein the analyte is an antigenic molecule.

8. The method of claim 7, wherein the antigenic molecule is insulin and the first affinity molecule is an antibody to insulin.

9. The method of claim 8, wherein the antibody to insulin is a monoclonal antibody.

10. The method of claim 9, wherein the second affinity molecule is a monoclonal antibody that binds to insulin at a different epitope than the first affinity molecule .

11. The method of claim 1, wherein the analyte is an antibody.

12. The method of claim 11, wherein the antibody is an insulin antibody and the first affinity molecule is selected from the group consisting of human insulin, bovine insulin, porcine insulin, and proinsulin.

13. The method of claim 12, wherein the labeled second affinity molecule is an antibody that specifically binds to the insulin antibody.

14. The method of claim 13, wherein the label for the second affinity molecule is selected from the group consisting of alkaline phosphatase, horse radish peroxidase, ╬▓-lactamase, ╬▓-galactosidase, and urease.

15. The method claim 11, wherein the antibody is a glutamic acid decarboxylase antibody.

16. The method of claim 11, wherein the antibody is a pyruvate dehydrogenase antibody.

17. The method of claim 1, wherein the analyte is a receptor molecule.

18. The method of claim 1, wherein the target molecule is biotin and the capture molecule is selected from the group consisting of avidin and strepavidin.

19. The method of claim 1, wherein the solid matrix is selected from the group consisting of culture dishes, beads, and membranes,

20. A kit for the detection of an analyte according to the method of claim 1 comprising a first affinity molecule having a specific affinity for the analyte; a target molecule; a spacer; a capture molecule; and a reporter system comprising a second affinity molecule having a specific affinity for the analyte, said second affinity molecule having a detectable label attached thereto.

21. The kit of claim 14, wherein the target molecule and the first affinity molecule are covalently attached via the spacer.

22. A method of quantitation of an analyte in a test solution comprising the steps of a) contacting the analyte with a first affinity molecule and a second affinity molecule to form a bound complex, wherein the first affinity molecule and the second affinity molecules bind to distinct epitopes of the analyte, wherein the first affinity molecule is covalently linked to a target molecule, wherein the second affinity molecule is labeled; b) immobilizing the bound complex to a solid matrix, said solid matrix being precoated with a capture molecule, wherein the capture molecule has a specific affinity for the target molecule; c) washing the solid matrix to remove unimmobilized materials; d) quantitating the amount of analyte in the sample from the amount of label immobilized.

23. A method for detecting in a biological fluid the presence of antibodies to at least two antigens selected from the group consisting of insulin, GAD and ICA-512 comprising the steps of: a) contacting the biological fluid with each of the at least two antigens to form a bound complex, wherein each antigen is covalently linked to a target molecule via a carbon spacer; b) further contacting the incubation mixture from a) with a solid matrix, said solid matrix being precoated with a capture molecule, wherein the capture molecule has a specific affinity for the target molecule; c) contacting the immobilized complex with an affinity molecule, wherein the affinity molecule has a detectable label and specifically binds to the antibodies. d) removing unbound affinity molecules; and e) quantitating the amount of antibodies in the sample from the amount of label immobilized.

24. The method of claim 23, wherein the carbon spacer is selected from the group consisting of straight chain carbon spacer, branched chain carbon spacer, and heterocyclic carbon spacer.

25. The method of claim 24, wherein the spacer is a straight chain carbon spacer and has a chain length of 3 to 20 carbon atoms.

26. The method of claim 25, wherein the spacer has a chain length of 13 carbon atoms .

27. The method of claim 23, wherein the detectable label on the affinity molecule is selected from the group consisting of enzyme label, radioactive label, and fluorescent label.

28. The method of claim 27, wherein the enzyme label is selected from the group consisting of alkaline phosphatase, horse radish peroxidase, ╬▓-lactamase, ╬▓- galactosidase, and urease .

29. The method of claim 23, wherein the target molecule is biotin and the capture molecule is selected from the group consisting of avidin and strepavidin.

30. The method of claim 23, wherein the solid matrix is selected from the group consisting of culture dishes, beads, and membranes,

31. The method of claim 23, wherein the at least two antigens are insulin and GAD.

32. The method of claim 23, wherein the at least two antigens are insulin and ICA-512.

33. The method of claim 23, wherein the at least two antigens are GAD and ICA-512.

34. The method of claim 23, wherein the at least two antigens are insulin, GAD and ICA-512.

35. A method for detecting in a biological fluid the presence of antibodies to at least two antigens selected from the group consisting of insulin, GAD and ICA-512 comprising the steps of: a) contacting the biological fluid with at least one of the at least two antigens to form a bound complex, wherein the at least one antigen is covalently linked to a target molecule via a carbon spacer; b) further contacting the incubation mixture from a) with a solid matrix, said solid matrix being precoated with a capture molecule, wherein the capture molecule has a specific affinity for the target molecule, and wherein the solid matrix is precoated with at least one of the at least two antigens, wherein the at least one of antigens precoated on the solid matrix is different from the at least one antigen covalently linked to the target molecule; c) contacting the immobilized complexes with an affinity molecule, wherein the affinity molecule has a detectable label and specifically binds to the antibodies; d) removing unbound labeled affinity molecules; and e) quantitating the amount of antibodies in the sample from the amount of label immobilized.

36. The method of claim 35, wherein the carbon spacer is selected from the group consisting of straight chain carbon spacer, branched chain carbon spacer, and heterocyclic carbon spacer.

37. The method of claim 36, wherein the spacer is a straight chain carbon spacer and has a chain length of 3 to 20 carbon atoms.

38. The method of claim 37, wherein the spacer has a chain length of 13 carbon atoms .

39. The method of claim 35, wherein the detectable label on the affinity molecule is selected from the group consisting of enzyme label, radioactive label, and fluorescent label .

40. The method of claim 39, wherein the enzyme label is selected from the group consisting of alkaline phosphatase, horse radish peroxidase, ╬▓-lactamase, ╬▓- galactosidase, and urease .

41. The method of claim 35, wherein the target molecule is biotin and the capture molecule is selected from the group consisting of avidin and strepavidin.

42. The method of claim 35, wherein the solid matrix is selected from the group consisting of culture dishes, beads, and membranes,

43. A kit for the detection of antibodies to insulin, GAD and ICA-512 comprising insulin, GAD and ICA-512; a target molecule; a spacer; a capture molecule; and a labeled affinity molecule having a specific affinity for antibodies to insulin, GAD and ICA-512.

44. The kit of claim 43 further comprising 6- histidine ICA-512 and a monoclonal antibody to 6- histidine ICA-512.

45. The kit of claim 43, wherein the target molecule and the first affinity molecule are covalently attached via the spacer.