Solid-Phase Enzyme-Immunoassay of Anti-Insulin Antibiodies : Effect of Labeling Site in Insulin and of Labeled Number of Horseradish Peroxidase on the Assay Sensitivity

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Three horseradish peroxidase (HRP)-labeled porcine insulins which have definite labeling site(s) were compared regarding sensitivity in a solid-phase enzyme-immunoassay (EIA) of anti-insulin antibodies. The standard curves obtained with Lys^B29-HRP-insulin and Gly^A1-HRP-insulin were steeper than that with Gly^A1, Lys^B29-diHRP-insulin for both polyclonal and monoclonal antibodies. Thus, the mono-HRP-labeled insulins can afford higher sensitivities in the EIA. The importance of the HRP-labeling site in insulin and of the number of labeled HRP was first demonstrated by using HRP-labeled insulins having definite labeling site(s).