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Clinical and Diagnostic Laboratory Immunology, May 2001, p. 482-488, Vol. 8, No. 3
1071-412X/01/$04.00+0   DOI: 10.1128/CDLI.8.3.482-488.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Development of a Novel In Vitro Assay (ALS Assay) for Evaluation of Vaccine-Induced Antibody Secretion from Circulating Mucosal Lymphocytes

H. Sunny Chang* and David A. Sackdagger

Vaccine Testing Unit, Department of International Health, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205

Received 24 April 2000/Returned for modification 4 December 2000/Accepted 17 January 2001

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% CO2 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.

* Corresponding author. Mailing address: 10326 Champions way, Laurel, MD 20723. Phone: (410) 955-7937. Fax: (301) 604-2076. E-mail: hhz123{at}yahoo.com

dagger Present address: ICDDR, Mohakhali, Dhaka 1000, Bangladesh.

Clinical and Diagnostic Laboratory Immunology, May 2001, p. 482-488, Vol. 8, No. 3
1071-412X/01/$04.00+0   DOI: 10.1128/CDLI.8.3.482-488.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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