Activation of human B lymphocytes. I. Direct plaque-forming cell assay for the measurement of polyclonal activation and antigenic stimulation of human B lymphocytes

A model for the detection of single cell antibody production by human tonsillar lymphocytes after stimulation with either sheep red blood cells (SRBC) or polyclonal B-cell activators has been described. The culture system is a modified Mishell-Dutton technique with certain critical factors identified. The assay is a sensitive and resproducible hemolysis-in-gel system employing an ultra-thin layer gel technique measuring plaque-forming cells (PFC) against SRBC targets. Several factors essential for optimal responses are described, but the critical feature of the culture system is the use of selected lots of human AB serum supplements which are extensively absorbed with SRBC. This removes a blocking factor present in most human serum which suppresses the B-cell response to SRBC targets after stimulation with either SRBC or several polyclonal B-cell activators. In addition, absorption of serum with SRBC eliminates the presence of artifactual plaques. Background PFC are extremely low and stimulated cultures show significant and reproducible responses. These studies provide a simple, sensitive, and reproducible model for probing the complex events associated with activation of human B lymphocytes.