The optimum conditions for antibody production in vitro by Peyer's patch lymphocytes and a comparison between total immunoglobulins and antigen (TNP)-specific antibody synthesis following mitogen stimulation

Abstract

Using optimised conditions in terms of cell concentration (2 X 10(6)/ml), duration of culture (7 days) and pH (7.0-7.1) and the highly sensitive enzyme-linked immunosorbent assay for antibody quantitation, we assessed the effects of lipopolysaccharide (LPS) and concanavalin A (Con A) on the levels of both total immunoglobulins and TNP-specific antibodies and compared the proportion of IgA anti-TNP to total IgA synthesised following the in vivo priming of mice, 5 days prior to culture, with trinitrophenylated sheep red blood cells (TNP-SRBC). LPS stimulation resulted in an increase in total IgA (ninefold), IgM (sixfold) and IgG (eightfold) synthesis over unstimulated cultures and similar increases were seen in IgA anti-TNP (eightfold), IgM anti-TNP (fivefold) and IgG anti-TNP (twelvefold). Con A stimulation resulted in a slight increase in total IgA (threefold), IgG (twofold) but a decrease in IgM (twofold) but did not appear to affect TNP-specific antibody levels. In unstimulated cultures, IgA anti-TNP comprised 7.8% of the total IgA synthesised. In LPS and Con A stimulated cultures the proportion was 6.6% and 3.2%, respectively. Inclusion of 10% fetal calf serum in the culture medium resulted in a substantial increase in the synthesis of IgG, IgM and IgA in unstimulated cultures but increased IgA levels only in LPS stimulated cultures. Our results emphasise the importance of optimising culture conditions when studying in vitro antibody synthesis and demonstrate that Peyer's patches respond quite substantially to parenterally administered antigen and that these responses can subsequently be monitored in vitro.