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.
The humoral immune response against sheep red blood cells (SRBC) is one of the most sensitive and frequently used end-points 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 micrograms 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Temple
L
L
Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond 23298.
Kawabata
T T
TT
Munson
A E
AE
White
K L
KL
D000818:Animals; D000910:Antibodies, Heterophile / Q000096:biosynthesis / Q000097:blood*; D001564:Benzo(a)pyrene / Q000494:pharmacology*; D003520:Cyclophosphamide / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000276:immunology*; D005260:Female; D006462:Hemolytic Plaque Technique; D007075:Immunoglobulin M / Q000096:biosynthesis / Q000097:blood; D051379:Mice; D008815:Mice, Inbred Strains; D051381:Rats; D011916:Rats, Inbred F344; D012756:Sheep / Q000276:immunology*
D000910:Antibodies, Heterophile; D007075:Immunoglobulin M; D001564:Benzo(a)pyrene; D003520:Cyclophosphamide