Fluorescence Enzyme Immunoassay for Insulin using Peroxidase-Tyramine-Hydrogen Peroxide

概要

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A method for the enzyme immunoassay of insulin is described. Insulin was conjugated with horseradish peroxidase by the periodate oxidation method. Separation of the bound and free fractions was accomplished by a double-antibody solid phase method using Sepharose 4B-anti-rabbit IgG goat IgG. The amount of bound enzyme-labelled insulin was determined by measuring the fluorescence developed after incubation with tyramine and hydrogen peroxide. Tyramine was a better fluorogenic substrate for the determination of peroxidase activity homovanilic acid. Insulin levels in serum could be measured over the range of 2.5 to 160 μU/ml, which is similar to that of insulin radioimmunoassay. Insulin values in 34 serum samples were determined using this method and RIA. A good correlation (γ=0.89) was obtained. The coefficient of variation was 0.6-2.6%. The method is roughly equivalent to RIA with respect to sensitivity. EIA largely overcomes the problems of RIA because there is no radiation hazard or disposal difficulties. Moreover, the laboratory equipment required is relatively inexpensive and readily available, while the label enzyme, HRP, is reasonably price and has a long shelf life. The enzyme immunoassay of insulin described in this paper should make it possible to perform routine assays even in laboratories with limited facilities.
公益社団法人日本薬学会の論文
1979-10-25