Simple method for development of sensitive and specific antiinsulin antisera for laboratory use.
Commercial sources provide good, though expensive antiinsulin antisera. We describe here a simple, fast and inexpensive method for the production of antiinsulin antisera. Purified pork insulin (Lente) was injected subcutaneously in oil/water/complete Freund adjuvant mixture. Three guinea pigs received 0.25 mg of insulin and three received 0.5 mg of insulin. Subsequent injections of the same dose were done 40 and 60 days later. Five animals developed antisera with titers superior to 1:10,000 40 days after the primary inoculation. Four out of five guinea pigs improved further their antibody titer after the 2nd and 3rd injection (p less than 0.0005). Good sensitivity was associated with titers superior to 1:50,000 and appeared only after the 2nd injection to improve further after the 3rd. Thus, four out of six animals developed antiinsulin antisera suitable for the radioimmunoassay (RIA). The antisera bound proinsulin on an equimolar basis with insulin while glucagon was not bound up to 100 ng/ml. The minimum detectable insulin concentration was about 12 pg (0.3 microU) at the optimum antiserum dilution. Six animals given a small dose of insulin (0.06 mg) developed antisera of a low titer and sensitivity, unsuitable for the RIA.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000917:Antibody Formation; D000918:Antibody Specificity; D005934:Glucagon / Q000276:immunology; D006168:Guinea Pigs; D007328:Insulin / Q000276:immunology; D049528:Insulin, Long-Acting / Q000276:immunology; D011384:Proinsulin / Q000276:immunology; D011863:Radioimmunoassay; D013552:Swine
D000906:Antibodies; D007328:Insulin; D049528:Insulin, Long-Acting; D005934:Glucagon; D011384:Proinsulin