Interlaboratory study of the primary antibody response to sheep red blood cells in outbred rodents following exposure to cyclophosphamide or dexamethasone

Abstract

EPA guidelines provide a choice in evaluating humoral immune system function in rats and mice immunized with sheep red blood cells (sRBC): an antibody-forming cell (AFC) assay or a sRBC-specific serum IgM enzyme-linked immunosorbent assay (ELISA). Four different laboratories used both methods to detect suppression of the antibody response by cyclophosphamide (CP) or dexamethasone (DEX). Attempts were made to minimize interlaboratory variability through the use of common reagents and vendors; each laboratory used the same source for rodents, immunosuppressive agents, and one sheep for sRBCs, and determined optimal sRBC concentration for immunization and peak day of antibody response in female CD rats and CD1 mice. The CP dose at which statistical significance was first observed in each species was quite similar within each lab using either assay. For DEX, the AFC assay detected significant and greater suppression at lower concentrations compared to the ELISA in both rats and mice. All labs detected DEX suppression using an AFC assay, whereas only one lab detected significant suppression in both species using an ELISA. For both compounds the magnitude of suppression was greater using the AFC assay, and resulted in ID(50) values which were lower in the AFC assay when compared to the ELISA. In addition, cross-species comparisons of ID(50) values suggested rats were more sensitive than mice. These initial experiments with two chemicals indicated that the AFC assay is consistently better at identifying suppression of a T-dependent antibody response across laboratories following xenobiotic exposures in outbred rats and mice. Additional compounds will need to be evaluated before concluding that one method is superior or more sensitive to the other in detecting suppression of the antibody response.