Use of Lysine (B29)-Horseradish Peroxidase Labeled Porcine Insulin in Solid Phase Antibody Enzyme-Immunoassay of Human Insulin

Abstract

Horseradish peroxidase (HRP)-labeled porcine insulins (GlyA1-HRP-insulin, Lys^B29-HRP-insulin and Gly^A1, Lys^B29-diHRP-insulin) have been evaluated for their efficiency in solidphase antibody competitive enzyme-immunoassay (EIA) of human insulin using a mouse anti-human insulin monoclonal antibody that recognizes the A8-A10 loop determinant of insulin. The amount of Lys^B29-HRP-insulin bound to the antibody coated on a polystyrene ball is greater than that of GlyA1-HRP-insulin and much greater than that of Gly^A1, Lys^B29-diHRP-insulin. Two EIA methods, by simultaneous additiion and delayed addition of HRPlabeled insulin, are described for the assay of insulin. Lys^B29-HRP-insulin and Gly^A1-HRPinsulin afforded higher sensitivity in detecting insulin than the diHRP insulin. Using Lys^B29-HRP-insulin, which provides the highest sensitivity, the detection limits for insulin are 1.0 and 0.1 μU, respectively, in the simultaneous and delayed addition method. HRP activity is measured fluorimetrically with 3-(p-hydroxyphenyl) propionic acid and hydrogen peroxide.