Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/575—Hormones
  • G01N2333/62—Insulins

Definitions

• 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) .
• IDDM Insulin Dependent Diabetes Mellitus
• 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 .
• bindable reactants are allowed to react with each other under conditions that facilitate binding.
• 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.
• two general approaches have been adopted.
• 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.
• 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) .
• detection techniques in the first category include the enzyme-linked immunoassays and the antibody sandwich assay.
• 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 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.
• solid support materials 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.
• 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.
• 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.
• Radioimmunoassays RIA
• fluorescent immunoassays FIA
• Radioimmunoassay technique requires the handling of radioactive materials and detection usually involves expensive equipment. Similarly, detection of fluorescence requires expensive and cumbersome equipment.
• soluble bindable molecules e.g. antigens
• affinity molecule e.g. antibody
• RIA the most commonly used method of quantitating insulin in test samples.
• a higher level of detection of insulin antibodies is achieved when the antibodies and insulin are allowed to react in a solution phase.
• 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.
• substrate bound assays like ELISAs and solution-phase assays like RIA
• RIA solution-phase assays
• 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.
• 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) .
• IDDM insulin- dependent diabetes mellitus
• NIDDM non-insulin dependent diabetes mellitus
• IDDM is an autoimmune disease targeting ⁇ -cells of the islets of Langerhans in the pancreas. What initiates the autoimmune response against ⁇ -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 ⁇ -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) .
• ICA islet cells
• IAA insulin
• GAD 65kD glutamic acid decarboxylase
• 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.
• ICA immunoprecipit
• 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.
• detector molecules or first affinity molecules
• the detector molecules and the test sample containing the analyte are allowed to react in a solution phase.
• 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.
• the mixture is exposed to a solid matrix precoated with capture molecules having a specific affinity for the target molecule.
• 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.
• 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.
• 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.
• 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.
• ligand molecules can be detected by using their specific receptors as the detector molecules .
• 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.
• the detector molecule is a ligand.
• 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.
• 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.
• 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.
• binding molecules 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 .
• 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.
• 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 .
• 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.
• 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 .
• 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.
• 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.
• 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.
• 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.
• capture molecule is meant for the purpose of specifications and claims, a molecule that exhibits specific affinity for the target molecule.
• the capture molecule may be a specific antibody, a non-immunological molecule (e.g. avidin or strepavidin), or a magnetic substance.
• detector molecule or "first affinity molecule” is meant for the purposes of specifications and claims, a molecule having a high affinity for the analyte.
• a detector molecule is one of the members of a bindable pair.
• 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.
• 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.
• 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.
• 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.
• 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.
• bound complexes are immobilized via attachment of the target molecule to a solid matrix that is precoated with the capture molecule.
• 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.
• the second affinity molecule binds to a different epitope of the analyte than the first affinity molecule i.e. the detector molecule.
• 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.
• the detector molecule is a peptide or a polypeptide
• 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 .
• 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.
• target molecule should not only have an affinity for the capture molecule but should also be covalently attachable to the detector molecules.
• suitable target molecule - capture molecule pairs include both immunological pairs (e.g. antigen-antibody) and nonimmmunological pairs (e.g. biotin-avidin) .
• 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.
• 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.
• spacer chain length 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 .
• PEG 600 polyethylene glycol
• the spacer may be straight or branched one. It is preferable to use a straight chain spacer of 3-18 carbons.
• 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.
• 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.
• 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.
• 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.
• Suitable blocking agents include proteins like albumins, gelatin, nonspecific IgG, nonfat dry milk, and surfactants like Tween 20, Tween 80, and Triton X-100.
• optimal binding conditions can be determined by techniques well to those skilled in the art. These include variation of buffer, pH, ionic conditions etc.
• 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, ⁇ - lactamase, ⁇ -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 .
• 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.
• 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.
• 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.
• 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.
• kits for a method for the detection of an analyte comprising 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.
• kits for a method for the detection of an antibody comprising 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.
• a detector molecule insulin, GAD and ICA-512
• a spacer a target molecule
• a capture molecule a labeled affinity molecule having a specific affinity for the antibody.
• a monoclonal antibodies for 6-His ICA-512, and 6-His ICA-512 are included.
• 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 .
• 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.
• 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.
• 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.
• 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.
• capture molecules in solution are added to the solid substrate .
• 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.
• 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.
• avidin was coated on to microtiter plates.
• the coating was achieved by passive adsorption in a suitable buffer.
• a buffer phosphate buffered saline, Sigma
• glycine buffered saline pH 8.6
• 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,
• Avidin coated plates can be stored in sterile PBS for up to one year and used when needed.
• This embodiment demonstrates that solution phase ELISA of the present invention is more sensitive than the conventional solid phase ELISA.
• detection of insulin antibodies in human serum samples was carried out .
• 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.
• each sample was run 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
• 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.
• 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.
• This embodiment illustrates the detection of antigens using solution phase ELISA of the present invention.
• 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.
• 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.
• serum insulin levels can be quantitated.
• 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.
• mABs 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 .
• 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.
• a labeled second antibody directed towards the test antibody e.g. anti-human IgG, if the test sample is human
• 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.
• 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.
• 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 ⁇ g/ml avidin. The wells were incubated for 30 minutes at room temperature, washed several times with a buffer (PBS) and incubated with 100 ⁇ l/well strepavidin- alkaline phosphatase (Jackson Labs, 1:500 dilution) for 30 minutes at room temperature.
• PBS buffer
• 100 ⁇ l/well strepavidin- alkaline phosphatase Jackson Labs, 1:500 dilution
• Biotinylated GAD 125ng 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.
• antibodies to the antigen ICA-512 are detected using the solution phase ELISA of the present invention.
• ICA-512 can be purified by methods known in the art.
• 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.
• 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.
• 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.
• 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) .
• detergents for example Triton X-100, PMSF, AEBSF, Leupeptin, and DNase
• 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) .
• PD10 columns molecular exclusion chromatography
• 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 ⁇ g/ml avidin. The wells are incubated for 30 minutes at room temperature, washed several times with a buffer (PBS) and incubated with 100 ⁇ l/well strepavidin-alkaline phosphatase (Jackson Labs, 1:500 dilution) for 30 minutes at room temperature.
• PBS buffer
• 100 ⁇ l/well strepavidin-alkaline phosphatase Jackson Labs, 1:500 dilution
• 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.
• 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.
• 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.
• a anti-6-His murine monoclonal antibody in carbonate buffer pH 9.6
• 50 ng of a anti-6-His murine monoclonal antibody in carbonate buffer pH 9.6
• 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.
• each of the three ELISAs for insulin, GAD and ICA-512
• test samples for example, serum samples
• biotin labeled insulin, GAD and ICA-512 either separately or together.
• the incubation mixture is then poured onto an avidin or strepavidin coated plate.
• bound complexes containing autoantibodies can be detected using a mixture of labeled antibodies directed to the autoantibodies.
• 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) .
• 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
• 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.
• second affinity molecule labeled second affinity molecule
• 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.
• 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.
• 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) .
• a labeled second antibody e.g. anti human IgG and IgM conjugated to alkaline phosphatase
• 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
• 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 .
• 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 ⁇ g/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.
• 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) .
• IMMCO Diagnostics immunofluorescence kit
• 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) .
• neurotransmitters e.g. dopamine, muscarinic and acetylcholine
• hormone receptors for both peptides and steroids
• ion channel proteins imbedded in cell membranes are also included in this category of receptor- like molecules.
• a ligand for the receptor is used as the detector molecule.
• the ligand can be attached to a target molecule via a spacer.
• 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.
• an enzyme- labeled antibody directed towards the receptor is added.
• 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.

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