EVIDENCE FOR THE INSULIN-DIRECTED SPECIFICITY OF RABBIT ANTI-INSULIN SERUM 1

Abstract

Intravenous injection of rabbits with crystalline beef insulin led to the appearance of antibodies which hemolyzed insulin-conjugated erythrocytes in the presence of complement. This lysis could be inhibited by preincubation of antibodies with crystalline insulin before the addition of the insulin conjugated red cells. The degree of inhibition varied with the amount of free insulin preincubated. Therefore, this method seemed applicable to the quantitative determination of crystalline insulin. However, the reliability of the method is dependent on the specificity of the antiserum employed. When egg albumin, human serum albumin, human gamma globulin, beef glucagon, bovine serum albumin and bovine gamma globulin were preincubated with the rabbit insulin antiserum, lysis was not inhibited appreciably. It appears, therefore, that proteins other than insulin, including 3 derived from the same species as the insulin used for immunization, did not react with the insulin antibodies. Highly purified samples of sheep, pork and beef insulin (prepared by counter-current diffusion) inhibited lysis to about the same extent as an equal amount of the insulin employed for immunization. These data indicate a high degree of reactivity between the beef insulin antibodies and sheep and pork insulins. They also suggest that the antibodies were specific for insulin and not impurities, unless these impurities were present in equal amounts in the various insulins. One hundred [mu]g of a crude beef pancreatic extract, containing about 1.8 [mu]g of insulin, and 1.8 [mu]ug of crystalline beef insulin were preincubated with 2 series of the same dilutions of rabbit antiserum. However, when the inhibition curves were compared it was found that inhibition due to the crude fraction was slightly less than that caused by the insulin. If the rabbit antiserum contained antibodies to antigens other than insulin, inhibition by the crude (containing 50 times more protein than the pure preparation) should have been much greater than that by the pure material. Various rabbit anti-insulin sera were tested in mice for their ability to block the hypoglycemic action of insulin. Mice were injected intraperitoneally either with a mixture of insulin and antiserum or insulin and normal rabbit serum. It was found that the antiserum prolonged the length of time the mice could maintain themselves on an inclined screen as compared to mice injected with the control mixture. These different types of evidence strengthen the conviction that the rabbit antiserum is highly specific for insulin.