부산대학교 도서관

The aim of the present study was to set up a sensitive technical alternative to the classical procedures for detection of human insulin antibodies. We developed a method of post-embedding immunoelectron microscopy (IEM) using as the substrate fresh human pancreas, embedded in acrylic resin to maintain its antigenic structure. The antigen was insulin within the mature secretory granule. Serum samples obtained from 10 patients with insulin antibodies detected at various titers by either radio binding assay (RBA) or enzymatic immunoassay (EIA) were incubated for 2 hr at 37 degrees C at dilutions of 1:25, 1:100, 1:400, 1:1600, 1:6400, 1:25,600, and 1:102,400. The electron microscope photographs were analyzed by computerized morphometry and the number of protein A-gold-IgG complexes was calculated per micron2 of insulin granule. IEM results were compared with those obtained with EIA. The specificity of both techniques towards insulin was assessed as the difference between the signals (number of gold particles per micron2 of insulin granule in IEM or optical density > or = 0.193 in EIA) with and without excess insulin. Sensitivity was defined as the detection limit of the assay. In all the 10 sera investigated, IEM was more sensitive, with a 12- to 40-fold lower detection limit than EIA. IEM, with native insulin granules as substrate, is a specific, reproducible, and sensitive method for detection of human serum insulin antibodies. These findings also suggest IEM as a procedure potentially suitable for identifying antigen specificity of autoantibodies circulating at low concentration.