mmunological Studies of Rickettsia Purification of Complement-fixing Antigens of Rickettsia orientalis by Ether Extraction

Abstract

Group (species)-specific soluble antigen and type (strain)-specific particle antigen of Rickettsia orientalis (Gilliam, Karp and Kato strains) were obtained simultaneously by ether extraction of yolk sac suspension previously treated with Amberlite XE-64.
The infected yolk sac suspension was partially purified by differential centrifugation and Amberlite XE-64 treatment, and then extracted with cold anaesthetic ether. The obtained aqueous layer was centrifuged at 10, 000rpm for 30min, and its supernatant fluids were concentrated by ultrafiltration with Visking tube until one fourth (Giliam and Karp) or one eighth (Kato) the weight of the original yolk sacs after dialysis with veronal. buffered saline (pH 7.3) for 18hrs. On the other hand, the sediments obtained from the auqueous layer by centrifugation were washed again with veronal buffered saline after that it was resuspended in a volume of veronal buffered saline equal to one fourth (Gilliam and Karp) or one eighth (Kato) the weight of the original yolk sacs.
The complement-fixing titers of soluble antigens thus obtained were 16-128 units in Gilliam strain and 16-64 units in Karp and Kato strains against homologous mouse antiserums. The titers of particle antigens were 8-32 units in Gilliam strain and 4-8 units in Karp and Kato strains.
As the results of cross complement fixation test with both soluble and particle antigens against mouse antiserums from each strain, soluble antigen was group (species)-specific and particle antigen showed to have type (strain) specificity.
The complement-fixing titer of soluble antigen was almost completely destroyed by heating at 80°C for 30 min, but particle antigen could not be inactivated by the exposure to 100°C for 30 min. Both soluble and particle antigens were not inactivated by trypsin and phosphatide acyl-hydrolase (phospholipase A, Cobra venom). Soluble antigen was partially inactivated by periodic oxidation, but particle antigen was resistant to periodate. No influence was observed with the complement-fixing titers of both antigens on lyophilization.