PRODUCTION OF AMONOCLONAL ANTIBODY BASE SANDWICH ASSAY

THE GOAL OF THE RESEARCH IS TO GENERATE DIAGNOSTIC REAGENTS AND/OR THERAPEUTIC REAGENTS FOR DIABETES THROUGH PRODUCTION OF MONOCLONAL ANTIBODIES TO THE PEPTIDE HORMONE, AMYLIN. AMYLIN IS A NEWLY DISCOVERED PEPTIDE HORMONE THAT IS ACTIVE IN THE CONTTROL OF GLUCOSE METABOLISM AND THE MEASUREMENT OF PEPTIDE LEVELS DIRECTLY IN PLASMA WILL BE KEY TO FURTHER UNDERSTANDING AND QUANTITATING THE ROLE THAT THE HORMONE PLAYS IN THE DISEASE PROCESSES OF TYPE I AND TYPE II DIABETES, MONOCLONAL ANTIBODY REAGENTS WILL I) ASSURE STABLE SUPPLIES OF HIGH AFFINITY ANTIBODIES SPECIFIC TO DIFFERENT EPITOPES ON THE AMYLIN PROTEIN, II) BE USED TO MAKE ISOTOPE OR ENZYME CONJUGATED ANTIBODIES SPECIFIC FOR AMYLIN AND, III) BE USEFUL FOR THE DEVELOPMENT OF A SANDWICH ASSAY FOR AMYLIN. DURING PHASE I, WE WILL CHARACTERIZE THE MONOCLONAL ANTIBODIES PRODUCED FOR: I) SPECIFICITY TO AMYLIN, II) APPARENT AFFINITY CONSTANT, III) EPITOPE MAPPING USING PEPTIDE FRAGMENTS, IV) ABILITY TO FORM A COMPLEX WITH OTHER MONOCLONAL ANTIBODIES AND/OR AVAILABLE POLYCLONAL ANTISERA, V) IMMUNOGLOBULIN SUBTYPE. IN PHASE II, WE WILL USE THESE REAGENTS TO I) DEVELOP A SANDWICH ASSAY FOR AMYLIN, II) CONSTRUCT AN AFFINITY COLUMN TO PURIFY AND FURTHER CHARACTERIZE AMYLIN FROM HUMAN PLASMA: III) IDENTIFY ANTIBODIES THAT BLOCK AMYLIN BINDING TO ITS RECEPTOR AND USE THESE ANTIBODIES TO MODULATE AMYLIN ACTION IN VIVO: IV) CHARACTERIZE THE METABOLIC PRODUCTS OF AMYLIN.