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

In the previous paper (2) a method was de-scribed 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. How-ever, the applicability of the method to the meas-urement 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 an-swers were sought to these questions: 1) To what extent will the insulin antisera react with contami-nants that may be present in the crystalline beef insulin used for immunization? 2) Will these anti-sera 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 Schultz-Dale 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 sen-sitization and complement fixation experiments. However, these experiments were qualitative in nature. Furthermore, the insulin preparation