Antigenicity of insulin

Rabbit antisera to either crystalline or amorphous beef insulin agglutinated tanned sheep red blood cells coated with amorphous insulin but not with a sample of crystalline insulin. In contrast, guinea pig antisera agglutinated red blood cells coated with either insulin preparation. Crystalline beef insulin altered by treatment with acid-alcohol at 56° C renders tanned sheep red blood cells agglutinable by rabbit as well as guinea pig antiserum. Crystalline beef insulin, amorphous beef insulin and altered beef insulin are equally capable of inhibiting the hemagglutination reaction of rabbit antisera with tanned red blood cells coated with altered or amorphous insulin. By acrylamide gel disc electrophoresis, crystalline insulin could be resolved into 5 components. The three fractions obtained in sufficient quantity to test all inhibited the reaction of rabbit antisera with altered beef insulin. Rabbits injected with beef insulin plus complete Freund adjuvant respond promptly with production of high titers of antibody to altered beef insulin. After 4 to 5 weeks, titers decrease sharply and cannot be restored by booster injections. In tanned cell hemagglutination tests, rabbit antisera to beef insulin cross-react strongly with sheep insulin but slightly, if at all, with pork insulin. Rabbit antisera to pork insulin react more strongly with beef or sheep insulins than with crystalline or altered pork insulin. However, in inhibition tests, pork insulin is as active as the equivalent beef or sheep preparations. Rabbit antisera to beef or pork insulins regularly cross-react with crude rabbit insulin. Rabbit insulin inhibited the reaction of some of these antisera with beef insulin.