Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of smooth-lipopolysaccharide heterogeneity among Brucella biovars related to A and M specificities.
Smooth (S)-lipopolysaccharide (LPS) preparations from reference and field strains of several biovars of Brucella abortus, B. melitensis, and B. suis were prepared by (i) the hot phenol-water method, (ii) hot sodium dodecyl sulfate extraction and proteinase K digestion, or (iii) dimethyl sulfoxide extraction. These S-LPS-enriched fractions were further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining after periodate oxidation. Immunoblots were developed by using either monoclonal antibodies specific for Brucella A or M antigens or polyclonal polyspecific or monospecific sera from rabbits, cattle, and goats. The specificity of monoclonal antibodies reactive with Brucella unique (A or M) epitopes was demonstrated by enzyme-linked immunosorbent assay, LPS latex agglutination, or agglutination inhibition. The most-represented subunits of S-LPS ranged in Mr from 30,000 to 70,000 relative to marker proteins. According to A or M immunodominance, two sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns were clearly distinguished among biovars, whatever the fraction tested: a close succession of regularly spaced narrow bands for A greater than M strains and regularly spaced triplets of bands including either (i) a first thin band followed by two thick bands for B. abortus M greater than A strains or (ii) one thick band between two thin bands for B. melitensis or B. suis M greater than A strains. Moreover, A and M specificities were reaffirmed by sandwich enzyme immunoassay and latex agglutination inhibition with monoclonal antibodies and polyclonal sera.
Garin-Bastuji
B
B
Laboratoire Central de Recherches Vétérinaires, Centre National d'Etudes Vétérinaries et Alimentaires, Maisons-Alfort, France.
Bowden
R A
RA
Dubray
G
G
Limet
J N
JN
D000818:Animals; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000302:isolation & purification; D015373:Bacterial Typing Techniques; D002002:Brucella / Q000032:analysis / Q000145:classification / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008070:Lipopolysaccharides / Q000276:immunology* / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D008070:Lipopolysaccharides; D012967:Sodium Dodecyl Sulfate