Rickettsial indirect hemagglutination test: isolation of erythrocyte-sensitizing substance.

The erythrocyte-sensitizing substances (ESS) of Rickettsia prowazekii and R. conorii were characterized by biological and chemical criteria. ESS could be derived from either soluble or particulate complement-fixing antigens obtained by ether extraction of rickettsiae. The soluble complement-fixing antigen exhibited two peaks of serological activity in potassium tartrate density gradients. The particulate complement-fixing antigen coincided with the more dense peak but was distinguishable by its sedimentation in rate-zonal sucrose gradients. ESS was obtained from each of the complement-fixing-reactive gradient peaks by extraction with hot alkali and was quantified by a modified indirect hemagglutination test. These ESS preparations sedimented similarly in potassium tartrate gradients and were shown to contain protein and carbohydrate, both by colorimetric tests and by incorporation of radioactive precursors. The serological activity of ESS was unaffected by trypsin, but both antigenicity and erythrocyte-binding capacity were reduced after exposure to sodium metaperiodate. Highly purified ESS was rapidly inactivated by potassium tartrate and required stabilization with bovine plasma albumin.