Immunochemical identification of antigens of Brucella melitensis by means of CRIE.

A crude extract of Brucella melitensis was obtained by sonication, centrifugation and dialysis, and analyzed by quantitative immunoelectrophoresis. CIE analysis, using a high titer purified rabbit antibody anti-brucella fraction, showed that the Brucella melitensis extract contained at least 22 antigens that all moved towards the anode. The antigens of the crude extract able to interact with IgG of patients affected by brucellosis, were identified by means of CRIE using sheep 125I-anti human IgG. Fifteen sera of patients affected by brucellosis were analyzed by CRIE and three (Ag6, Ag12, Ag15) of the 22 antigens were able to bind specific human IgG to their corresponding immunoprecipitates. Ag6 was the only one present after one day of the CRIE plate exposure. After treatment of brucellosis patients with antibiotics for two weeks, the three antigens were still able to bind the specific IgG, after two months of treatment the antigens were reduced to two (Ag6, Ag15), while after ten months only one antigen (Ag6) was still able to bind specific human IgG. The brucellosis titer determined with common methods gave normal values after ten months of therapy. These results suggest that Ag6 is the most important antigen specific for human IgG. A preliminary determination of the molecular weight range of these antigens was carried out by a combination of size exclusion chromatography and CIE analysis. The Kav values were all above 66 kD.