Assay for anti-INGAP antibodies

Abstract

A solid phase assay is used for detecting antibodies to INGAP 104-118 peptide, a 15-amino acid peptide that is the biologically active portion of islet neogenesis associated protein (INGAP). The isotype of the antibodies to INGAP 104-118 peptide can be determined. A kit can also be used in the detection of anti-INGAP 104-118 antibodies. Endogenous autoantibodies or antibody production during therapeutic treatment of a mammal with INGAP 104-118 can be monitored.

1 . A method for detecting antibodies to INGAP 104-118  peptide in a test sample and determining isotype of said antibodies, comprising: 
 contacting a test sample from a human with INGAP 104-118  peptide bound to a solid support, wherein the contacting is under conditions sufficient for binding an anti-INGAP 104-118  antibody to the INGAP 104-118  peptide;    contacting the solid support with an isotype-specific antibody which specifically binds antibody molecules of one isotype of the human; and    determining the isotype-specific antibody bound to the solid support, wherein detection antibody bound to the solid support indicates that the test sample contains antibodies to INGAP 104-118  peptide and that the antibodies to INGAP 104-118  peptide are of the one isotype; wherein the test sample is selected from the group consisting of blood, serum and plasma.    
     
     
         2 . The method of  claim 1  wherein the isotype-specific antibody comprises a detectable label.  
     
     
         3 . The method of  claim 2  wherein the detectable label is an enzyme.  
     
     
         4 . The method of  claim 3  wherein the enzyme is selected from the group consisting of alkaline phosphatase, horseradish peroxidase, and beta-galactosidase.  
     
     
         5 . The method of  claim 2  wherein the detectable label is selected from the group consisting of fluorescent molecules, chemiluminescent molecules, radioactive molecules, and dye molecules.  
     
     
         6 . A kit for detecting an anti-INGAP 104-118  antibody in a test sample of a mammal comprising: 
 an INGAP 104-118  peptide; and    a detection antibody.    
     
     
         7 . The kit of  claim 6  wherein the test sample is selected from the group consisting of blood, serum and plasma.  
     
     
         8 . The kit of  claim 6  wherein the detection antibody specifically binds antibody molecules of all isotypes of a mammal.  
     
     
         9 . The kit of  claim 6  wherein the detection antibody is an isotype-specific antibody which specifically binds antibody molecules of one isotype of a mammal.  
     
     
         10 . The kit of  claim 6  wherein the detection antibody is detectably labeled.  
     
     
         11 . The kit of  claim 6  wherein the INGAP 104-118  peptide is bound to a solid support.  
     
     
         12 . The kit of  claim 6  further comprising an anti-INGAP 104-118  antibody.  
     
     
         13 . The kit of  claim 6  further comprising instructions for detecting antibodies to INGAP 104-118  peptide using the peptide and the antibody.  
     
     
         14 . The kit of  claim 6  further comprising a standard curve for use in calculating amount of anti-INGAP 104-118  antibody in a test sample.  
     
     
         15 . The kit of  claim 6  further comprising a buffer for washing unbound reagents.  
     
     
         16 . The kit of  claim 10  wherein the detectable label is selected from the group consisting of fluorescent molecules, chemiluminescent molecules, radioactive molecules, and dye molecules.  
     
     
         17 . A method for detecting neutralizing antibodies in a patient undergoing treatment with an Islet Neogenesis Associated Protein, comprising: 
 collecting a test sample from a patient being administered a therapeutic protein selected from the group comprising sequences 104-118 of SEQ ID NO 2, sequences 1-175 of SEQ ID No 2 or fragments thereof,    contacting the test sample with peptide corresponding to the therapeutic protein bound to a solid support under conditions sufficient for binding an antibody to the therapeutics protein,    contacting any test sample bound to the therapeutic protein on the solid support with a detection antibody which specifically binds to antibody molecules of all isotypes of the mammal;    washing the solid substrate to remove any unbound detection antibody molecules, and    determining the presence of detection antibody bound to the solid support, wherein the presence of any detection antibody bound to the solid support indicates that the test sample contains neutralizing antibodies to the therapeutic protein.    
     
     
         18 . The method of  claim 17  wherein amount of the detection antibody bound is compared to a standard curve generated using known amounts of an antibody to the therapeutic peptide.  
     
     
         19 . The method of  claim 17  wherein multiple test samples are used which are serial dilutions of the test sample of the mammal wherein the serial dilutions show relative measure of titre in a test sample or between test samples from different sources.  
     
     
         20 . The method of  claim 19  further comprising: determining a highest serial dilution at which a signal is generated, said highest serial dilution being the test sample used to evaluate titer of anti-INGAP 104-118  peptide antibodies in the mammal.  
     
     
         21 . The method of  claim 20  wherein the highest serial dilution of a first mammalian test sample is compared to a highest serial dilution of a second mammalian test sample to provide a comparative measure of titer in the first and second mammalian test samples.  
     
     
         22 . The method of  claim 17  wherein the detection antibody comprises a detectable label.  
     
     
         23 . The method of  claim 22  wherein the detectable label is an enzyme.  
     
     
         24 . The method of  claim 23  wherein the enzyme is selected from the group consisting of alkaline phosphatase, horseradish peroxidase and beta-galactosidase.  
     
     
         25 . The method of  claim 25  wherein the detectable label is selected from the group consisting of fluorescent molecules, chemiluminescent molecules, radioactive molecules, and dye molecules.