Immunoelectrophoretic Identification of Guinea Pig Anti-Insulin Antibodies

Abstract

Two anti-insulin antibodies from guinea pig antisera were isolated by chromatography on diethylaminoethyl cellulose (2) and examined by immunoelectrophoresis and by radioautography with the aid of I¹³¹-insulin and the rabbit antiserum against guinea pig serum components. Peak I or Peak II antibody fraction showed a single radioactive are in the γ-globulin (I) or in the β₂-globulin (II) region, respectively, indicating their difference in the electrophoretic mobility. When the original antisera were used, the resultant radioactive are represented a composite of the two arcs obtained with two isolated antibody fractions. The anode end of the Peak I antibody are coalesced with the middle portion of the Peak II antibody are, thus producing a single long line covering the β₂- and γ-globulin regions with a distint spur between these two areas. This line and the spur corresponded exactly with the long strong are (so-called γ-globulin arc) and with the very faint spur observed on the stained slide. The relative intensity of radioactive line in γ- and β₂-globulin portions roughly reflected the relative antibody activity in Peak I and Peak II antibody fractions. The method is extremely sensitive for detecting anti-insulin antibodies. Positive results were obtained with antibody concentrations as low as 0.033 to 0.16 µg/ml. In addition, it requires only a few microliters of the sample and the two different antibodies are clearly distinguishable. Two antibodies are different in their reaction with rabbit antiserum against guinea pig serum. Both antibodies have some antigenic determinants in common, but Peak II antibody has some determinants which are not present in Peak I antibody. The complexes prepared by incubating I¹³¹-insulin with the antisera migrate significantly faster than the free antibodies.