Antibody Elicited by Brucella Abortus Antigen in Vitro: Prolonged Production by Cell Suspensions

Abstract

Summary It has been found that Brucella antibody can be elicited in vitro. Spleen cell suspensions from rabbits immunized 6 to 13 months earlier to Brucella abortus antigen were antigenically stimulated in vitro and cultured up to 30 days. Four to five days after initiation of the response antibody-synthesizing cells could be detected by passive localized hemolysis in agar. For detecting the cells, the ratio of Brucella erythrocyte-sensitizing substance to erythrocytes proved critical. After the exponential phase of their appearance, at 4 to 6 days, plaque-forming cells could no longer be demonstrated. The Brucella-stimulated cells synthesized high concentrations of specific bacterial agglutinins. Extracellular antibody reached a maximum by 8 to 11 days and remained at this level for 1 to 2 weeks. Titers of 1:160 and 1:320 were attained regularly. The prolonged “steady-state” of antibody production permitted an examination of the late phase of the immune response in vitro and the effect of various factors on it. Within limits, the antibody response of the cultured spleen cells was a function of antigen concentration. Although 106 brucellae elicited a high level of antibody from 107 spleen cells, antibody synthesis was sustained for 1 to 2 weeks only when higher numbers of brucellae were present. The amount of antibody produced depended markedly on the normal rabbit serum used in the medium, the atmosphere, and the physical conditions of culture. It was found that actinomycin D at 0.1 to 1.0 µg/ml completely inhibited the secondary immune response of cultured cells to Brucella abortus antigen when added at 0 hr or up to day 3. Aflatoxin at 1 µg/ml reduced the response significantly when the agent was added up to day 3. On disc electrophoresis, the antibody elicited in vitro migrated as a single band with the slow γ globulins, at the same rate as Brucella antibody produced in the rabbit. On the basis of sensitivity to 0.1 M 2-mercaptoethanol, over 90% of the antibody was IgM. It was concluded that the prolonged production of antibody provided a sensitive means of studying the effect of various factors on the secondary immune response in vitro. With cultures of dispersed cells initiated at 107 spleen cells/ml, antigenically stimulated with 108 killed Brucella abortus and grown under rather rigid conditions, the results were reproducible.