Biochemical and biological properties of soluble protein preparations from Brucella abortus

Abstract

Soluble salt-extractable protein antigens (CSP) from Brucella abortus may be of potential value as a vaccine and as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. These protein antigens, excluding protoplasmic proteins, are those present in the matrix of the outer membrane and in the periplasmic space of gram-negative bacteria. Our research deals mainly with the proteins of the periplasmic space and the nonporin proteins of the outer membrane. We have isolated a group of proteins from intact inactivated cells using mild, nondestructive extraction procedures. The proteins thus obtained range in molecular weight from 10 000 to 51 000 daltons as determined under denaturing conditions and from 10 000 to 124 000 daltons under nondenaturing conditions. The isoelectric pH of these proteins range from 3.5 to 10 with numerous bands in the pH 4.5 to 5.5 region, and a few bands in the pH 6 to 10 region. The low pH region also contains those protein bands which can be labeled with the membrane impermeant reagent, diazotized [125I]-iodosulfanilic acid. The soluble protein preparations are antigenic in guinea pigs, rabbits and cattle, and immunogenic in lemmings, guinea pigs and cattle. Cross-reacting protein antigens are present in protein preparations from B. abortus strains 19 and 2308 when tested with heterologous rabbit sera and with sera from vaccinated and experimentally infected cattle. The soluble protein antigens can be rendered more immunogenic by chemical modification of the primary amino groups with acyl anhydrides, especially with dodecanoyl anhydride. Immunization with dodecanoyl--modified proteins resulted in decreased humoral antibody levels (as measured in guinea pigs and cattle) without loss of protective activity, as measured in lemmings. Furthermore, the soluble proteins have proven to be a sensitive, stable and reliable reagent in the enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Brucella abortus in serum of vaccinated and experimentally infected cattle. Other soluble protein antigen preparations have also been evaluated and compared with strain 19 proteins for immunogenic properties and for use as an ELISA reagent.