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© 1993 Oxford University Press

research-article

Comparison of ELISA and Plaque-Forming Cell Assays for Measuring the Humoral Immune Response to SRBC in Rats and Mice Treated with Benzo[a]pyrene or Cyclophosphamide

L. TEMPLE*,1, T. T. KAWABATA{dagger}, A. E. MUNSON* and K. L. WHITE, JR.*,{ddagger}

*Department of Pharmacology and Toxicology Richmond, Virginia 23298 {dagger}Procter and Gamble Co. Cincinnati, Ohio {ddagger}Biomedical Engineering Program, Virginia Commonwealth University Richmond, Virginia 23298

Received January 28, 1993; accepted July 21, 1993

Comparison of ELISA and Plaque-Forming Cell Assays for Measuring the Humoral Immune Response to SRBC in Rats and Mice Treated with Benzo[a]pyrene or Cyclophosphamide. TEMPLE, L., KAWABATA, T. T., MUNSON, A. E., AND WHITE, K. L., JR. (1993). Fundam. Appl. Toxicol. 21, 412–419.

The humoral immune response against sheep red blood cells (SRBC) is one of the most sensitive and frequently used endpoints in evaluating the immunotoxicity of drugs and chemicals in experimental animals. Currently, most immunotoxicology studies measure the SRBC IgM antibody response by quantitating the number of SRBC-specific IgM antibody-forming cells using the hemolytic plaque assay. On the other hand, measurement of serum SRBC-specific IgM could be an easier, more cost effective endpoint in evaluating the SRBC antibody response in rodents. A validated method to measure SRBC-specific IgM, however, has not been developed. Thus, the objectives of the studies presented were to develop and validate an enzyme-linked immunosorbent assay (ELISA) for SRBC-specific IgM. Hemoglobin-free, detergent-solubilized membrane preparations were chosen as antigen for the ELISA. Various sources of SRBC were found to be equally useful, and as little as 0.1 µg of protein per well was optimal. Kinetic studies of the IgM response showed the peak day to be on Day 5 (mice) and Day 6 (rats). To validate the usefulness of the method for immunotoxicologic studies, serum SRBC-specific IgM levels and number of SRBC-specific plaque-forming cells were compared in rats and mice treated with two well-characterized immunosuppressive agents: benzo[a]pyrene and cyclophosphamide. Administration of these chemicals was found to produce very similar dose-dependent decreases in serum SRBC IgM and IgM-specific plaque-forming cells. These two endpoints were equally sensitive to the effects of the immunosuppressive drugs. Based on the present findings and the sensitivity, simplicity, and potential for automation of the ELISA for SRBC-specific IgM, the ELISA could potentially replace the plaque assay as the first step in testing for potential immunotoxic chemicals.


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