Structural differences among antibodies of different specificities.

Abstract

Both the variety of specific serological reactions, and the chemical heterogeneity of the immune globulins suggest that differences in the specificity of antibodies are accompanied by appropriate differences in molecular structure. Speculation on the nature of relevant structural differences has led to two alternative assumptions: either antibody molecules of different specificity have identical primary structure and are folded differently,' or these antibody molecules have different amino acid sequences.' Up to the present, however, antibodies of the same size class but of different specificity have not conclusively been shown to differ significantly from each other, or from nonspecific y-globulin, in any pertinent structural characteristic. Recently, it has been found3 4 that 7-globulin molecules consist of several polypeptide chains that may be dissociated by cleavage of disulfide bonds. The possibility arose that this structural feature might be related to immunologic specificity. If this were so, the dissociated chains of different antibodies might show different patterns when appropriately separated. Starch gel electrophoresis in urea of reduced alkylated derivatives of y-globulin4 and purified antibodies was employed to investigate this possibility. In this study, the guinea pig was chosen as a source of antibodies. This animal species could be immunized conveniently, and. guinea pig -y-globulin, unlike rabbit 7y-globulin,4 could be easily dissociated by reduction. Antibodies from single animals were studied, in order to avoid the heterogeneity that might have resulted from pooling antisera. A number of antibodies directed against artificial haptens, and some antibodies specific for protein antigens, were compared by means of starch gel electrophoresis both before and after reduction and alkylation. Distinct differences were found in the electrophoretic patterns of reduced and alkylated specifically purified antibodies that were directed against different antigens. Materials and Methods.-(a) Animals: Guinea pigs of either sex and of heterologous stock, each weighing 500 gm or more were used. Pre-immunization bleedings were taken in order to compare the total -y-globulin with specific antibody. (b) Antigens: The following immunizing antigens were used: (1) Picryl-guinea pig albumin (Pic-GPA), preparations I, II, and III, with 28, 12, and 43 haptenic groups/mole of protein, respectively. (2) Picryl-ovalbumin (Pic-Ova), 15 groups/mole. (3) 2,4-dinitrophenyl-bovine ay-globulin (DNP-BGG), 7 groups/mole. (4) para-toluenesulfonyl-ovalbumin, (Tosyl-Ova), 22 groups/mole. (5) para-toluenesulfonyl-guinea pig albumin (Tosyl-GPA). (6) para-arsanilicdiazo-guinea pig albumin (As-GPA). (7) para-arsanilic diazo-bovine serum albumin, (As-BSA). (Antigens 5, 6, and 7 were not assayed for the number of haptenic groups conjugated to each mole of protein.) (8) Ovalbumin (Ova), 2 X recrystallized, Worthington Biochemical Corp., Freehold, N. J. (9) Bovine serum albumin (BSA), Armour Pharmaceutical Co., Kankakee, Illinois. (c) Other proteins: Guinea pig albumin was prepared from pooled sera, and guinea pig ,yglobulin from individual sera, by zone electrophoresis on starch.5 Bovine fibrinogen was obtained from Armour Pharmaceutical Co., Kankakee, Illinois. Protein concentrations of the -y-globulins were determined by the modified Folin-Ciocalteu method.6