Fractionation of Antihorse Sera against Diphtheria Toxin and Pertussis Bacillus by Salting-out and Electrophoresis

Abstract

Antisera against diphtheria toxin and pertussis bacillus were fractionated by salting-out at various concentrations of sodium sulfate. Supernatants and precipitates were separated by filtration. The precipitates were washed with the same concentrations of sodium sulfate solution, dialysed against running water for 24 hours and against the phosphate buffer used for the electrophoresis for 16 hours. The precipitates thus treated and the supernatants were analysed by electrophoresis.The antibody titer of the diphtheria antitoxin was determined by the flocculation test and the intracutaneous reaction in rabbits, while the antibody titer of the pertussis antisera was estimated by the agglutination test.A prominent peak, named T by Tiselius, was observed by the electrophoresis of the diphtheria antiserum, and a large γ2-peak in pertussis antiserum. According to the results of the fractionation experiments, the diphtheria antitoxin appears to be found mainly in T component, while the pertussis agglutinin in γ2-globulin. The former was precipitated almost completely at 18g/dl concentration of sodium sulfate but very little at 12g/dl, while the latter was precipitated apparently at 12g/dl and completelv at 16g/dl.Thus the physicochemical properties of antibody melecules, that is, the solubility in concentrated salt solution and the electrophoretic mobility, were found to differ strikingly in different kinds of antigen.