Studies in immunochemistry. 13. Preparation and properties of the ‘O’ somatic antigen of Shigella dysenteriae (Shiga)

Abstract

Acetone-dried cells of Shigella dysenteriae (Shiga) were extracted with diethylene glycol and a prepn. of the somatic "O" antigen was recovered. The material was purified by fractionation with acetone and with ammonium sulfate. The phospholipid component was removed from the antigenic material and the resulting polysaccharide-protein complex fractionated by high-speed centrifugation. A heavy, water-insoluble material was recovered at 25000 g; this was strongly antigenic. Un-combined, undegraded, specific polysaccharide was present in the original diethylene glycol extracts of Sh. dysenteriae. and was not deposited after prolonged centrifugation at 105000 g. The antigen fractions separating between 25000 and 105000 g, contained 4.5-4.6% N and 0.80-85% P, were free from detectable impurities as shown by fractional solubility tests, electrophoretic examination and behavior in the analytical ultracentrifuge at pH 7.0. Specific polysaccharide dissociates progressively from the antigen at alkaline pH values. Sedimentation constants at pH''s 4.5 and 8 obtained for the phospho-lipid-free antigen and extrapolated to infinite dilution give approx. the same figure (0.007), which indicates a particle weight of the order of 1 x 107. The material possesses an LD50 of 80[mu]g. in the mouse and induces formation of "Shiga" agglutinins in rabbits on the intraven. injn. of 1 [mu]g.