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Assay for anti-ingap antibodiesRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Assay In Which An Enzyme Present Is A Label, Heterogeneous Or Solid Phase Assay System (e.g., Elisa, Etc.)Assay for anti-ingap antibodies description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070184504, Assay for anti-ingap antibodies. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application is a Divisional Application of parent application Ser. No. 10/376,046, filed on Feb. 27, 2003, which claims the benefit of provisional application Ser. No. 60/361,040 filed on Mar. 1, 2002. FIELD OF THE INVENTION [0002] The invention relates to the field of assays for antibodies. Specifically, the invention relates to antibodies raised in mammals against administered therapeutic agents. BACKGROUND OF THE INVENTION [0003] Pancreatic islets of Langerhans are the only organ of insulin production in the body. However, they have a limited capacity for regeneration. This limited regeneration capacity predisposes mammals to develop diabetes mellitus. Islet neogenesis associated protein (INGAP) plays a role in stimulation of islet neogenesis, in particular, in beta cell regeneration from ductal cells. INGAP.sup.104-118 peptide (IGLHDPSHGTLPNGS, SEQ ID NO: 1), a 15-amino acid peptide comprising amino acids 104-118 of the INGAP protein, is biologically active and is capable of inducing islet cell regeneration in an animal model. Pharmaceutical compositions containing a mammalian INGAP.sup.104-118 peptide can be used for treatment of endocrine pancreatic insufficiency which may result from diabetes mellitus. [0004] Antibodies to INGAP.sup.104-118 peptide may be generated in patients following repeated dosing of INGAP.sup.104-118 peptide or may be generated as autoantibodies to the endogenous protein, which may mitigate the action of INGAP or serve as a diagnostic marker for diabetes. Thus, there is a need in the art for a convenient assay for detecting antibodies that may be raised in a subject following treatment with INGAP.sup.104-118 peptide. BRIEF SUMMARY OF THE INVENTION [0005] One embodiment of the invention is a method for detecting antibodies to INGAP.sup.104-118 peptide in a test sample. This method comprises contacting a test sample which comprises serum of a mammal with INGAP.sup.104-118 peptide bound to a solid support. The contacting is done under conditions sufficient for binding an anti-INGAP.sup.104-118 antibody to the INGAP.sup.104-118 peptide. The solid support is contacted with a detection antibody which specifically binds antibody molecules of all isotypes of the mammal. The detection antibody bound to the solid support is determined. Detection antibody bound to the solid support indicates that the test sample contains antibodies to INGAP.sup.104-118 peptide. [0006] A second embodiment of the invention is a method for detecting antibodies to INGAP.sup.104-118 peptide in a test sample and determining the isotype of said antibodies. This method comprises contacting a test sample which comprises serum of a mammal with INGAP.sup.104-118 bound to a solid support. The contacting is done under conditions sufficient for binding an anti-INGAP.sup.104-118 antibody to the INGAP.sup.104-118 peptide. The solid support is contacted with an isotype-specific antibody which specifically binds antibody molecules of one isotype of the mammal. The isotype specific antibody bound to the solid support is determined. Detection antibody bound to the solid support indicates that the test sample contains antibodies to INGAP.sup.104-118 peptide and that the antibodies to the INGAP.sup.104-118 peptide are of the one isotype. [0007] A further embodiment of the invention is a kit for detecting an anti-INGAP.sup.104-118 antibody in the serum of a mammal. The kit comprises an INGAP.sup.104-118 peptide and a detection antibody. [0008] These and other embodiments of the invention provide the art with tools and methods for detecting antibodies that bind to INGAP.sup.104-118 peptide which are useful for monitoring subjects during treatment with INGAP.sup.104-118 peptide and identifying subjects with INGAP autoantibodies. BRIEF DESCRIPTION OF THE DRAWINGS [0009] FIG. 1. Exemplary schematic of anti-INGAP.sup.104-118 direct detection assay. Following the irreversible coating of peptide to the base of microtiter wells (1) test sample is added to wells (2). If antibodies specific for the peptide are present in the test sample they bind to the peptide and are retained in the wells after washing steps. The antibodies in contact with the peptide are detected by tagged-secondary antibodies which are subsequently added to the wells (3). Washing steps remove tagged-antibodies which are not in contact with the antibody-peptide complex. The presence of peptide-bound antibody is determined by reading the tag-signal in the wells (4). [0010] FIG. 2. Specificity of the rabbit antibody generated against INGAP.sup.104-118 peptide. Rabbit anti-INGAP.sup.104-118 peptide was incubated in microtiter wells pre-coated with either INGAP.sup.104-118 peptide, bovine serum albumin (BSA), INGAP.sup.151-164 (Cterm), or INGAP.sup.139-152 (Cseq). All wells were coated with an equivalent concentration of protein. Anti-INGAP.sup.104-118 antibody was detected using alkaline phosphatase-conjugated, anti-rabbit IgG in combination with p-nitrophenylphosphate and optical detection of p-nitrophenol at 405 nm. The horizontal line indicates the limit of background signal for the assay. [0011] FIG. 3. Sensitivity of the rabbit antibody generated to INGAP.sup.104-118 peptide. Rabbit anti-INGAP.sup.104-118 antibody was incubated in microtiter wells pre-coated with various concentrations of INGAP.sup.104-118 peptide. Detection of bound rabbit anti-INGAP.sup.104-118 antibody was monitored using alkaline phosphatase-conjugated, anti-rabbit IgG in combination with p-nitrophenylphosphate and optical detection of p-nitrophenol at 405 nm. The EC.sub.50 was 150 pg and the lowest detectable amount which was significantly different from zero was 18 pg. [0012] FIG. 4. Effect of human serum on detection of anti-INGAP.sup.104-118 peptide antibody. (A) Each data point represents 2-1000 ng/mL anti-INGAP.sup.104-118 antibody and 0 (.diamond-solid.), 5 (.box-solid.), 10 (.tangle-solidup.), 15 (.times.), 30 (*), or 50 (.circle-solid.) % normal human serum in microtiter wells. Detection of bound rabbit anti-INGAP.sup.104-118 antibody was monitored using alkaline phosphatase-conjugated, anti-rabbit IgG in combination with p-nitrophenylphosphate and optical detection of p-nitrophenol at 405 nm. (B) Reaction was carried out as in (A), but no rabbit anti-INGAP.sup.104-118 antibody was added. Data points correspond to 0 (.diamond-solid.), 5 (.tangle-solidup.), 10 (*), 15 (+), 30 (-), and 50% (.circle-solid.) normal human serum. [0013] FIG. 5. Anti-INGAP.sup.104-118 antibody assay sample plate layout. (1) Dilutions of patient serum samples. (2) Dilutions of standardized human sera. (3) Rabbit anti-INGAP.sup.104-118 antibody standard curve. (4) Plate blanks prepared using highest concentration of rabbit anti-INGAP.sup.104-118 antibody on wells not coated with INGAP.sup.104-118 peptide. DETAILED DESCRIPTION OF THE INVENTION [0014] It is a discovery of the present invention that anti-INGAP antibodies generated in vivo can be sensitively and specifically detected in a solid phase assay. Anti-INGAP.sup.104-118 antibodies can be detected without interference by the components of mammalian serum. Normal human serum does not contain factors that result in false positive signals, nor does it inhibit the interaction of anti-INGAP.sup.104-118 antibodies with INGAP.sup.104-118 peptide. [0015] Assays of anti-INGAP.sup.104-118 antibodies in a sample from a mammal are useful to monitor generation of neutralizing antibodies during the therapeutic administration of INGAP.sup.104-118 peptide. Neutralizing antibodies may be endogenous autoantibodies or antibodies generated in subjects following single or repeated dosing with INGAP.sup.104-118 peptide. Anti-INGAP.sup.104-118 antibodies can be specifically detected with sensitivity. [0016] In a solid phase assay, purified INGAP.sup.104-118 peptide can be immobilized on a solid support. Solid phase immunoassays are convenient for ease of separating bound from unbound components. Sequential immunoassay steps, including rinsing between steps and the binding of the detection antibody and development of the indicator reaction, can be easily performed without the need for expensive automation and skill. [0017] Any test sample can be used, including but not limited to blood, plasma, or serum. The invention particularly contemplates test samples containing serum. Serum can be obtained from any mammal including, for example, mouse, rat, rabbit, guinea pig, monkey, dog, cat, cow, goat, pig, and human. [0018] Test samples can be assayed at a single concentration of serum or at multiple concentrations which may be obtained by serial dilution of the serum. Diluent serum can be derived from the same species that is the source of the test sample. Other diluents such as buffers and normal saline can also be used. The highest serial dilution at which a signal can be detected can also be used to characterize a test sample. For example, the highest serial dilution at which a signal can be detected for a first mammal can be compared to that of a second mammal to obtain a relative measure of antibody titer in the first and second mammals. [0019] Any immunoreactive form of INGAP.sup.104-118, INGAP or a derivative thereof can be used. Native, synthetic, or recombinant forms of the whole INGAP peptide, or related proteins which contain, or are modified to contain the INGAP.sup.104-118 sequence or portions immunoreactive with an antibody against INGAP.sup.104-118 peptide may be used. Thus, portions of INGAP.sup.104-118 peptide or peptides containing such portions and other residues or moieties can be used. Derivatives include, but are not limited to, modification the peptide's C-terminus, N-terminus, and/or amino acid side chains. Examples of C-terminal carboxylate modifications include esterification (e.g., benzyl, methyl or ethyl ester) and amidation. Examples of N-terminal modifications include acetylation and alkoxycarbonylation. Amino acid side chain modifications include methylation, benzylation, t-butylation, tosylation, alkoxycarbonylation, and the like. Continue reading about Assay for anti-ingap antibodies... 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