Chromatographic studies of sera from calves vaccinated with brucella abortus, strain 19.

The presence of antibody was detected by agglutination tests in the serum of calves four days after vaccination with Brucella abortus strain 19. Titres had reached a maximum by seven to ten days post-vaccination. Sucrose density-gradient ultracentrifugation demonstrated that the earliest antibodies were macroglobulins, IgM (19Sgamma; gammaM)-globulins. Lighter antibodies, IgG (7Sgamma(2); gammaG)-globulins, appeared a few days later. With time, antibody titres fell, IgM declining somewhat more quickly than IgG. After revaccination some seven months later, there was a rapid rise in both IgM and IgG.Anion-exchange column chromatography (DEAE-cellulose) and gel filtration (Sephadex G-200) were applied in separating the two forms of antibody. The former method, in which a gradient buffer system was used, proved to be the more efficient; the IgG antibodies apeared in early eluates at pH 7.8 to 8.0 and low ionic strength, 0.03M, whereas IgM was eluted late when the pH had fallen below 6.0 and the molarity had increased to beyond 0.2. DEAE cellulose chromatography detected IgG as well as IgM sera collected as early as five days after vaccination.