Characterization of an immuno-dominant 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 reacted with Omp31, while only 11 reacted with Omp25, suggesting that Omp31 is identical to the previously reported immuno-dominant 29-kDa protein. Attempts to purify Omp31 on a larger scale by using procedures such as ion exchange-, reversed phase-, affinity- and gel filtration chromatography suggested that the outer membrane proteins were aggregated with rough lipopolysaccharide. Only denaturing SDS-gel filtration chromatography was able to separate proteins of about 29 kDa from rough lipopolysaccharide but did not separate Omp31 from Omp25 in B. ovis preparations. When used in an enzyme-linked immunosorbent assay, this 29-kDa protein preparation was less sensitive and less specific than the routinely used heat-extracted B. ovis antigen. A readily available recombinant E. coli, expressing the gene for Omp31 from Brucella melitensis 16 M, was used to extract and enrich recombinant Omp31 by a temperature-dependent Triton X-114-based technique. When this material was used in immunoblots with the 45 sera from B. ovis-infected sheep and with 10 monoclonal antibodies, raised against B. ovis Omp31, major differences in the antibody reactivity between the recombinant B. melitensis Omp31 and the B. ovis Omp31 were found. Such differences were unexpected because of the known structural and immunological relatedness of outer membrane proteins from various Brucella species. These results indicated that the antibody-response in B. ovis naturally-infected sheep against the immuno-dominant Omp31 was directed against epitopes which were only accessible when the protein was aggregated with rough lipopolysaccharides, or which were formed after aggregation but were not present in the recombinant protein.
Kittelberger
R
R
Central Animal Health Laboratory, Wallaceville Animal Research Centre, Upper Hutt, New Zealand. kittelbergerr@wallaceville.mqm.govt.nz
Diack
D S
DS
Vizcaíno
N
N
Zygmunt
M S
MS
Cloeckaert
A
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western / Q000662:veterinary; D002002:Brucella / Q000276:immunology*; D002006:Brucellosis / Q000175:diagnosis / Q000276:immunology / Q000662:veterinary; D002850:Chromatography, Gel / Q000662:veterinary; D002852:Chromatography, Ion Exchange / Q000662:veterinary; D003168:Complement Fixation Tests / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary*; D004823:Epididymitis / Q000175:diagnosis / Q000276:immunology / Q000662:veterinary; D004926:Escherichia coli / Q000737:chemistry; D005779:Immunodiffusion / Q000662:veterinary; D016056:Immunodominant Epitopes / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis / Q000276:immunology
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D016056:Immunodominant Epitopes; C101108:Omp31 protein, Brucella melitensis; D011994:Recombinant Proteins