Development of a novel in vitro assay (ALS assay) for evaluation of vaccine-induced antibody secretion from circulating mucosal lymphocytes

We describe here a novel method for measuring in vitro antibody secretion f rom the tissue culture of human B lymphocytes in peripheral blood mononucle ar cells (PBMC) after oral vaccination with a killed cholera vaccine. Enzym e-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 immu noglobulin A [IgA] and Igc) for up to 4 days of incubation at 37 degreesC w ith 5% CO2 in cell cultures, The secreted antibody concentration correlated positively with the PBMC concentration and incubation time in the tissue c ulture and correlated negatively with the storage time of the whole blood a t room temperature. In vitro assay of secreting antibody in the lymphocyte supernatant (i.e., the ALS assay) is capable of the detecting specific anti body response after oral vaccination with a killed whole-cell-plus-B-subuni t cholera vaccine (WC-B) in healthy adults in a phase I clinical trial. Pos timmunization PBMC secreted antibodies to cholera toxin in the cell superna tants, Antibody production did not require any in vitro antigen stimulation , In the ALS assay, antigen-specific antibody titers of prevaccination samp les were barely detectable, whereas serum antitoxin ELISA titers in backgro und 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 t he PBMC culture in postvaccination blood samples.