Genus-specific antigens in Leptospira revealed by immunoblotting

Immunoblotting of leptospiral sonicates with heterologous rabbit antisera revealed a distinct cross-reactive pattern which differed with respect to the pathogenic and non-pathogenic leptospiral serovars, and that all serovars tested from Leptospira interrogans, L. biflexa and L. illini contained a common 35 kilodalton (Kd) band. A leptospiral genus-specific antigen preparation produced by ethanol fractionation of L. biflexa serovar patoc reacted by enzyme immunoassay (EIA) with all heterologous serovars tested. Further purification using Sephacryl S-300 gel filtration revealed one major cross-reactive peak and several homologous peaks detectable by EIA. Gel electrophoresis of this peak revealed 3 major protein bands of 35, 34 and 29 Kd by Coomassie blue staining. This peak was further fractionated by high pressure liquid chromatography (HPLC), yielding 7 fractions, one of which cross-reacted. Rabbit antisera to this S-300/HPLC fraction reacted with all serovars tested. Immunoblotting revealed 2 distinct groups of cross-reactive antigens, a 33-35 Kd group that was proteinase K sensitive but not reduced by periodate oxidation, and a 14.4-26.5 Kd group whose activity was reduced by periodate but not proteinase K, indicating the presence of both protein and carbohydrate genus antigens. Immunoblotting L. interrogans serovar pomona flagella with S-300/HPLC antiserum suggested that the 35 Kd band found in all serovars tested was a flagellar component.