Evidence for the insulin-directed specificity of rabbit anti-insulin serum.

In the previous paper (2) a method was described for the immunological assay of microgram quantities of insulin. This method employed the reaction between rabbit antiserum to insulin and sheep erythrocytes conjugated with insulin. However, the applicability of the method to the measurement of insulin is obviously dependent upon the insulin-directed specificity of the antiserum which is employed in the assay. Therefore, the present experiments were designed to determine the specificity of rabbit antisera to crystalline beef insulin. In order to establish this specificity answers were sought to these questions: 1) To what extent will the insulin antisera react with contaminants that may be present in the crystalline beef insulin used for immunization? 2) Will these antisera react with proteins derived from beef plasma and other sources? 3) To what extent will the antisera to beef insulin react with insulin from other species? 4) Will the antisera interfere with the physiological action of insulin? Previous investigators have provided much information on some of these points. Lewis (3) and Barral and Roux (4) have shown that purified insulin is a different antigen from those in serum or crude extracts of pancreas. The immunological similarity of insulin prepared from several species has been demonstrated by Lewis (3) and also by Wasserman and Mirsky (5). Using the SchultzDale technique, Lewis (3) showed that guinea pigs can be sensitized with insulin from one species and shocked with insulin from another species. Wasserman and Mirsky (5) confirmed this in sensitization and complement fixation experiments. However, these experiments were qualitative in nature. Furthermore, the insulin preparations