Limiting dilution analysis of specific in vitro anti-herpes simplex virus antibody production by human lymphocytes

An enzyme linked immunosorbent assay (ELISA) system to study specific anti-herpes simplex virus (HSV) antibody production in vitro by human peripheral blood mononuclear cells (PBMC) has been developed. HSV specific antibody production was detected in culture supernatants of PBMC from HSV seropositive healthy individuals by stimulating with optimal concentrations of HSV antigen without mitogens. To investigate the specificity of the resulting antibody, a limiting dilution analysis was performed as follows. PBMC from HSV-1 seropositive and HSV-2 seronegative individuals were stimulated by wild type HSV-1 (KOS), glycoprotein C deletion mutant of HSV-1 (MP), and wild type HSV-2 (186), and the precursor frequencies of B cells which produce antibodies to HSV-1 (KOS), HSV-1 (MP) and HSV-2 (186) were then estimated. When PBMC from five individuals were stimulated by HSV-1 (KOS), the estimated precursor frequencies of B cells which produced antibodies to HSV-1 (KOS), HSV-1 (MP), and HSV-2 (186) were 1/79,000 to 1/37,000, 1/104,000 to 1/52,000. and 1/78,000 to 1/101,000, respectively. On the other hand, when PBMC were stimulated by HSV-1 (MP) and HSV-2 (186), the precursor frequencies of B cells which produced antibody to HSV-1 (KOS) were the same as those to HSV-1 (MP) and HSV-2 (186), respectively. By using the experimental systems reported here, it became possible to determine clearly the immune status to HSV type-specific and type-common antigens and to each viral glycoprotein.