Unit

UNIT 3.8 Assays for B Lymphocyte Function

  1. Subbarao Bondada,
  2. Darrell A. Robertson

Published Online: 1 NOV 2003

DOI: 10.1002/0471142735.im0308s56

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Bondada, S. and Robertson, D. A. 2003. Assays for B Lymphocyte Function. Current Protocols in Immunology. 56:II:3.8:3.8.1–3.8.24.

Author Information

  1. University of Kentucky, Lexington, Kentucky

Publication History

  1. Published Online: 1 NOV 2003
  2. Published Print: AUG 2003

Abstract

This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first basic protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. Alternatively, the number of antibody-producing cells can be quantified by plaque-forming cell (PFC) assays presented in this unit: the Cunningham-Szenberg and the Jerne-Nordin techniques. Both methods employ specially prepared slide chambers, described here, in which the antibody-producing B cells are mixed with complement and indicator sheep red blood cells (SRBC), or with trinitrophenol-modified SRBC (TNP-SRBC), with subsequent lysis and counting of plaques. Because IgM antibodies fix complement efficiently, whereas IgG and IgA antibodies do not, unmodified PFC assays measure only IgM antibodies. The assay can be modified, however, to measure all classes of antibodies or to enumerate total immunoglobulin-secreting B cells, as described in alternate protocols. Yet another method of measuring the number of antibody-producing B cells (in a class-specific fashion) is to use the ELISPOT technique described in UNIT 7.14. The resting B cells used in these procedures are prepared as described in the final support protocols for Percoll gradient centrifugation.