Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of smooth-lipopolysaccharide heterogeneity among Brucella biovars related to A and M specificities - PubMed (original) (raw)

B Garin-Bastuji et al. J Clin Microbiol. 1990 Oct.

Abstract

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.

PubMed Disclaimer

References

    1. Infect Immun. 1989 Sep;57(9):2820-8 -PubMed
    1. Infect Immun. 1970 Feb;1(2):174-82 -PubMed
    1. Infect Immun. 1989 Sep;57(9):2829-36 -PubMed
    1. Nature. 1970 Aug 15;227(5259):680-5 -PubMed
    1. Eur J Biochem. 1969 Jun;9(2):245-9 -PubMed

MeSH terms

Substances

LinkOut - more resources