Production of human antibodies to bee venom phospholipase A2 in vitro

Phospholipase A2 (PLA) is the major antigen of bee venom. Whereas individuals frequently stung by bees, such as bee keepers, show high levels of IgG4 anti-PLA antibodies in serum, most patients sensitive to bee venom possess increased IgE anti-PLA. We have established a culture system by which anti-PLA antibodies can be induced in vitro. Peripheral blood mononuclear cells were stimulated in a first step with PLA and/or pokeweed mitogen (PWM). After 3 days of culture the cells were washed and further incubated with fresh medium. Anti-PLA antibodies were estimated by an enzyme-linked immunosorbent assay (ELISA) and anti-PLA antibody secreting cells were counted by means of an ELISA plaque assay. Cells from bee keepers, but not those from normal donors, produced anti-PLA IgG in vitro. The isotype pattern of anti-PLA antibodies produced in vitro was identical to that found in serum of the respective donor. Anti-PLA IgM antibody-secreting cells developed at low frequencies in both bee keepers and normal donors whereas PLA-specific IgE could not be detected. The formation of anti-PLA IgG was suppressed by high amounts of antigen, while the IgM response was not affected. We conclude that PWM-induced antibodies reflect the in vivo situation of the donors and that anti-PLA IgG and IgM are regulated by different pathways and mechanisms.