A new method of evaluating the heterogeneity of anti-insulin antibodies in sera from insulintreated diabetic patients is described. Sera from five insulin-treated diabetic subjects were treated with acid charcoal to remove endogeneous and/or exogeneous insulin, followed by neutralization of pH and incubation with 125I-insulin. After incubation with 125I-'nsulin, each serum sample was mixed with dextran-coated charcoal (DCC) solution in saline. With constant stirring, pH of the DCC solution was decreased stepwise, and dissociated 125I-insulin from antibodies was adsorbed to DCC. The heterogeneity of anti-insulin antibodies regarding their binding ability of insulin was quantitatively expressed as 4pH90 20.that is, the pH span between 90% and 20% binding of 125I-insulitno the antibodie.s The relatives trength of antibodies against acid treatment is expressed as pH50%, that is, the pH value in which 50% of the bound 125I-insulinw as dissociated from the antibodies.The values of 4pH90 20 and pH50% varied from 0.84 to 1.89 and from 6.14 to 6.62 respectively. Because the number of the sera investigated was small, the relationships between these parameters of anti-insulin antibodies and the duration and dose of insulin treatment are not clear at present. Further accumulations of data will clarify these relationships.
In order to compare the extraction methods of IRG from the tissues by the acid ethanol and by the acid saline methods, the rat submaxillary gland and pancreas and the autopsied human pancreas were extracted by these two methods and IRG was determined using antiglucagon serum OAL-123. In the acid ethanol method, Kenny's extraction and the direct assay method of acid ethanol extract were compared. Rat and human pancreata contained large amounts of IRG in direct assay of acid ethanol extract and a little less in Kenny's extraction, but, in the acid saline extract, little IRG was found in both types of pancreas. On the contrary, the rat submaxillary gland contained a large amount of IRG in acid saline extract and little in acid ethanol extract, including Kenny's method. These findings suggest that a different extraction method would have to be used according to the tissues in some cases, even when the same immunoreactive hormone is to be extracted, or that the submaxillary gland might contain some other glucagon-related substances.