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

In competitive enzyme-immunoassays (EIAs) of insulin, mixtures of several forms of enzyme-labeled insulins prepared using glutaraldehyde1), maleimide2) or periodate 3,4) have been used . There is no detailed comparison of enzyme-labeled insulins with respect to the sensitivity of EIA of insulin. The enzyme-labeling site (s) and the number of enzyme molecules incorporated per insulin molecule may be important factors influencing the sensitivity of the EIA. Recently we have developed a method for the preparation of horseradish peroxidase (HRP ; EC 1. 11. 1. 7) -labeled porcine insulins (HRP-insulins ; GlyA1-HRPinsulin, LysB29-HRP-insulin and GlyA1, LysB29-diHRP-insulin)5) (Fig. 1). This paper surveys the immunoreactivity of the above HRP-insulins with a mouse anti-human insulin monoclonal antibody and assesses the sensitivity of each as competitors in solid-phase EIA of human insulin. Two EIA methods for human insnlin are examined : a rapid method by simultaneous addition of HRPinsulin and a highly sensitive method by delayed addition of HRP-insulin.