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

Abstract

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 degrees 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.

FIG. 1

Plot of the average cell counts of three human normal blood samples, as determined by a hemocytometer, which were taken with citrate anticoagulant and stored at 25°C. A 10-ml portion of each sample was processed for PBMC isolation at 0, 24, 48, and 72 h.

FIG. 2

Plot of T-cell proliferation after ConA stimulation in blood samples obtained from human volunteers. Three blood samples from healthy volunteers were collected (no vaccination) and stored at 25°C. These samples were processed at day 0, day 1, day 2, and day 3, and the cell concentrations were adjusted to 10⁶ cells/ml. The cells were harvested and count to determine H³ incorporation. The results are expressed as the ratio of sample counts with ConA to the corresponding control counts obtained without ConA.

FIG. 3

Residual scatter plot for the regression model showing predictive value versus residuals.