CHEMILUMINESCENCE IMMUNOASSAY FOR INSULIN

THE AIM OF THIS PROJECT IS TO DEMONSTRATE THE FEASIBILITY OF AN IMMUNOASSAY FOR INSULIN, USING ACRIDINIUM ESTERS AS A CHEMILUMINESCENT LABEL. THIS WILL LEAD TO A TECHNIQUE FOR THE DETECTION OF POLYPEPTIDE HORMONES THAT IS FASTER AND MORE SENSITIVE, AND REMOVES THE NEED FOR (125)I-LABELED PEPTIDES OR ANTIBODIES. THE PROJECT WILL EXPLORE BOTH TRADITIONAL IMMUNOASSAYS IN WHICH THE INSULIN MOLECULE IS LABELED AND "SANDWICH"-TYPE ASSAYS IN WHICH INSULIN ANTIBODIES ARE USED. IN THE FIRST CASE, LABELED INSULIN WILL BE PURIFIED BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) AND DIRECTLY SUBSTITUTED AS THETRACER IN A RADIOIMMUNOASSAY (RIA) PROCEDURE. FOR THE IMMUNOMETRIC ASSAY, ANTIBODIES TO INSULIN WILL BE LABELED AND PURIFIED BY GEL FILTRATION CHROMATOGRAPHY. THE LABELED PEPTIDE AND ANTIBODY WILL BE COMPARED FOR STABILITY AGAINST A COMMERCIALLY AVAILABLE RIA USING (125)I-LABELED INSULIN. THE ASSAY WILL SERVE AS A MODEL FOROTHER PEPTIDE HORMONE ASSAYS THAT REQUIRE HIGH SENSITIVITY AND STABLE REAGENTS. THE PROJECT WILL INVOLVE LIMITED ASSAY DEVELOPMENT, COMPARISON WITH A COMMERCIAL RIA, AND A LIMITED TRIAL WITH PLASMA SAMPLES. THE MULTIMILLION-DOLLAR HORMONE RIA MARKET MIGHT INCREASE IFMORE STABLE, SAFER, CHEAPER, AND SIMPLER ASSAYS WERE AVAILABLE USING CHEMILUMINESCENCE DETECTION TECHNIQUES.