CHARACTERIZATION OF AN IMMUNODOMINANT ANTIGEN IN BRUCELLA-OVIS AND EVALUATION OF ITS USE IN AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY

A panel of 45 Brucella ovis serologically positive sera were tested in immunoblots against B. ovis outer membrane proteins Omp31 and Omp25, purified by preparative SDS-gel electrophoresis. Forty-three sera reac ted with Omp31, while only 11 reacted with Omp25, suggesting that Omp3 1 is identical to the previously reported immune-dominant 29-kDa prote in, Attempts to purify Omp31 on a larger scale by using procedures suc h as ion exchange-, reversed phase-, affinity- and gel filtration chro matography suggested that the outer membrane proteins were aggregated with rough lipopolysaccharide. Only denaturing SDS-gel filtration chro matography was able to separate proteins of about 29 kDa from rough Li popolysaccharide but did not separate Omp31 from Omp25 in B. ovis prep arations. When used in an enzyme-linked immunosorbent assay, this 29-k Da protein preparation was less sensitive and less specific than the r outinely used heat-extracted B. ovis antigen. A readily available reco mbinant E. coli, expressing the gene for Omp31 from Brucella melitensi s 16 M, was used to extract and enrich recombinant Omp31 by a temperat ure-dependent Triton X-114-based technique. When this material was use d in immunoblots with the 45 sera from B. ovis-infected sheep and with 10 monoclonal antibodies, raised against B. ovis Omp31, major differe nces in the antibody reactivity between the recombinant B. melitensis Omp31 and the B, ovis Omp31 were found. Such differences were unexpect ed because of the known structural and immunological relatedness of ou ter membrane proteins from various Brucella species. These results ind icated that the antibody-response in B. ovis naturally-infected sheep against the immune-dominant Omp31 was directed against epitopes which were only accessible when the protein was aggregated with rough lipopo lysaccharides, or which were formed after aggregation but were not pre sent in the recombinant protein. (C) 1998 Elsevier Science B.V.