We describe here a novel method for measuring in vitro antibody secretion from the tissue culture of human B lymphocytes in peripheral blood mononuclear cells (PBMC) after oral vaccination with a killed cholera vaccine. Enzyme-linked immunosorbent assay (ELISA) titers of the antibody secreted in the cell supernatant were determined. The validation results demonstrated that human PBMC remained viable and continued to secrete antibodies (total immunoglobulin A [IgA] and IgG) for up to 4 days of incubation at 37°C with 5% CO 2 in cell cultures. The secreted antibody concentration correlated positively with the PBMC concentration and incubation time in the tissue culture and correlated negatively with the storage time of the whole blood at room temperature. In vitro assay of secreting antibody in the lymphocyte supernatant (i.e., the ALS assay) is capable of the detecting specific antibody response after oral vaccination with a killed whole-cell-plus-B-subunit cholera vaccine (WC-B) in healthy adults in a phase I clinical trial. Postimmunization PBMC secreted antibodies to cholera toxin in the cell supernatants. Antibody production did not require any in vitro antigen stimulation. In the ALS assay, antigen-specific antibody titers of prevaccination samples were barely detectable, whereas serum antitoxin ELISA titers in background of prevaccine samples were significantly higher than the ALS titers. We conclude that, without any in vitro antigen stimulation after vaccination, PBMC secrete antibodies into the supernatants in the ALS assay. This assay can quantitatively measure the antigen-specific antibody production from the PBMC culture in postvaccination blood samples.Postvaccination immunity is generally assessed via the use of antibodies in serum, but it is impossible to distinguish between recently produced antibodies and preexisting antibodies. Antibody levels in serum do not represent the latest immune responses accurately, because serum antibodies include the accumulated soluble antibodies that were induced by previous exposure to antigens.Recent antigen exposure of mucosal T and B cells induces proliferation and differentiation of these cells (14,25). The activated T and B cells circulate through the thoracic duct into the blood and eventually return to common mucosal sites, such as the lamina propria of the intestine, as matured plasma cells (2,17,20,22,23,26).To develop a sensitive surrogate for assaying local immunity, the lymphocytes traveling from local mucosal areas to the systemic blood circulation are used by methods for in vitro laboratory evaluations such as ELISPOT (6-10, 12, 15, 21; P. W. Lowry, L. M. McFarland, and H. K. Threefoot, Letter, J. Infect. Dis. 154:730, 1986). In its final step, ELISPOT measures the results of specific antibody-secreting cells (ASC) on a spot-forming gel (11)(12)(13)15,18; Lowry et al., letter). ELISPOT measures the number of antibody producing cells per 10 6 PBMC following oral vaccination (11,16). The quantification of antibodies secreted by a fixed concentration of ...