Extraction of membrane antigens from Brucella ovis and an assessment of their serological activity by immunoblotting.
The efficacy and selectivity of chaotropic and phase-partitioning procedures for the extraction of membrane proteins from Brucella ovis were compared with a standard Sarkosyl method. Major group 1, 2 and 3 outer-membrane proteins (OMPs) of B. ovis stained by Coomassie blue in SDS-PAGE gels had, respectively, apparent molecular masses of 81/82 kDa, 39-41 kDa and 30-32 kDa. The presence of these bands in the Sarkosyl extract of total membrane vesicles (TMVs) indicate that the procedure failed to selectively solubilize only inner-membrane proteins (IMPs). SDS-PAGE analyses also revealed the presence of OMPs and other additional bands following extraction of B. ovis TMVs by butanol phase-partitioning or with extraction solutions based on the chaotropic reagents potassium thiocyanate (KSCN), sodium salicylate (SSC) and lithium acetate (LAE). OMPs are therefore not selectively extracted by any one of these procedures. Based on the number and staining intensity of extracted membrane-associated polypeptides, the efficacy of different extraction procedures could be graded in decreasing order as follows: KSCN, SSC, butanol and LAE. Both butanol and SSC were particularly effective in extracting group 3 OMPs. Sera from chronic excretor rams were used to identify zones of seroreactivity in immunoblots. Essentially, two reactivity patterns were seen: strong antibody binding against polypeptides in zones A (46-85 kDa), C (28-32 kDa) and D (18-22 kDa) in one, and additional reactivity against zones B (34-44 kDa) and E (13-18 kDa) polypeptides in the other.(ABSTRACT TRUNCATED AT 250 WORDS)
Chin
J
J
Immunology Section, Elizabeth Macarthur Agricultural Institute, New South Wales Agriculture and Fisheries, Camden, Australia.
Turner
B
B
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D000942:Antigens, Bacterial / Q000032:analysis*; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology / Q000302:isolation & purification*; D015153:Blotting, Western; D002002:Brucella / Q000276:immunology*; D002006:Brucellosis / Q000276:immunology; D000440:Butanols; D002462:Cell Membrane / Q000737:chemistry / Q000276:immunology; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D012756:Sheep
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D000440:Butanols