Immunopurified extracellular Bartonella henselae antigen for detecting specific antibodies by enzyme immunoassay

Protein antigens of Bartonella henselae bacterial sonicate supernatant and concentrated cell-free culture filtrate were examined by SDS-PAGE. The sonicate supernatant gave 38 bands and the culture filtrate at least 21, of which 18 were of bacterial origin. Immunoblotting against 13 monoclonal antibodies obtained from mice infected with live B. henselae showed that 10 of these antibodies reacted with a narrow 225 kDa band and varying smears of bands ranging from 36 to 240 kDa in the sonicate, but only with a single 200 kDa band in the culture filtrate. Testing of pre-and post-infection rabbit sera in immunoblotting against culture filtrate demonstrated that the 200 kDa component gave the most prominent specific reaction with post-infection sera. The 200 kDa antigen was isolated by immunoaffinity chromatography of concentrated culture filtrate, and its molecular size determined by size-exclusion chromatography as >1000 kDa. The immunopurified antigen was compared with bacterial sonicate as coating antigen in EIA for determining humoral immune responses in rabbits inoculated with live B. henselae. The two antigens pave almost identical results for IgM and IgG responses. The specificity of the immunopurified antigen was tested in EIA against hyperimmune rabbit sera and sera of rabbits inoculated with live B. henselae, B. quintana and Afipia felis. Only the hyperimmune serum against B. henselae and the sera of the rabbits inoculated with live B. henselae reacted with the immunopurified antigen, whereas the B. henselae sonicate cross-reacted with hyperimmune and postinfection sera of rabbits inoculated with B. quintana and A, felis.