Comparison of Primary Immune Responses to SRBC and KLH in Rodents

Abstract

In rodents, the Plaque Assay, T-dependent antibody response to sheep erythrocytes (SRBC), has been reported to be a sensitive and predictive functional immune assay for detecting immunomodulatory compounds. However, various laboratories have chosen to use ELISA-based assays for evaluating the primary immune response in rodents. The ELISA-based assays offer several advantages over the Plaque Assay, which make them attractive for use in immunotoxicological evaluations. Among the most popular antigens used in the ELISA-based assays are SRBC and more recently KLH. While the Plaque Assay and the ELISA-based assays are both capable of evaluating the humoral immune response, they are measuring different endpoints. The Plaque Assay focuses primarily on splenic effects. ELISA-based assays, which use serum from immunized animals, are holistic in nature in that these assays measure effects of antibody production on the spleen, lymph nodes, and bone marrow. Depending on the drug or compound evaluated, different effects and degrees of sensitivity can be seen with the Plaque Assay and ELISA-based assays. One recent finding is that the sensitizing dose of KLH used in the KLH ELISA differentially affects the responses observed in rodents. Even within the same species, different strains of mice and rats produce different magnitudes of responses to the same sensitizing dose. A key component of this discussion focuses on the sensitivity of the Plaque Assay as compared to KLH ELISA-based assays. These assays were evaluated by comparing the response obtained following administration of several known immunosuppressive agents, including cyclophosphamide, azathioprine, cyclosporine A and dexamethasone. The effects on the primary IgM immune response in the B 6 C 3 F 1 mice, the primary immunotoxicological rodents used by National Toxicology Program, and in the Sprague-Dawley rat, the primary rodent models used by industry are addressed.

Evaluation of primary and secondary responses to a T-cell-dependent antigen, keyhole limpet hemocyanin, in rats

“…Traditional primary SRBC plaque responses have been reported to be more sensitive to detect the immunosuppressive effects of CPA and cyclosporine A than the primary KLH-ELISA responses in rodents (White et al, 2007). In the present study, CPA inhibited both KLH and SRBC-induced primary antibody responses at the same dose in either assay system.…”

“…In fact, two immunosuppressive compounds, e.g., 2-methoxyethanol and 2-methoxy-acetic acid, have shown different susceptibility in the PFC assay among rat strains (Smialowicz et al, 1994). Similar strain differences in anti-KLH IgM responses have been reported to exist in a KLH-ELISA-based TDAR assay using mice and rats (White et al, 2007). In addition, the inherent inter-individual variability of the antibody responses-especially in the out-bred strains-has been assumed to be affected by variations in analytical methods, i.e., ELISA (Gore, 2006).…”

“…The differences in the sensitivity of TDAR methodologies have also long been a topic for discussion (Herzyk and Holsapple, 2007;White et al, 2007). Traditional primary SRBC plaque responses have been reported to be more sensitive to detect the immunosuppressive effects of CPA and cyclosporine A than the primary KLH-ELISA responses in rodents (White et al, 2007).…”

“…The dose levels were set at 0 (vehicle control), 1, 3, or 6 mg CPA/kg/day, based on a preliminary study (data not shown). An intravenous (IV) dose of KLH-saline solution delivering 300 µg KLH antigen/rat was selected based on the earlier studies of Gore et al (2004) and White et al (2007). The results of the preliminary study which included a time-course response revealed that IV dosing of 300 µg KLH/rat produced sufficient levels of anti-KLH specific antibody that were, in turn, considered to be utilizable for detecting the immunosuppressive potential of the test articles, even in the absence of any other overt toxicity findings.…”

A Sub Acute Immunotoxicity Study in G?ttingen Minipigs? with the Immunosuppressive Compounds Cyclosporin A and Dexamethasone

“…The absence of a (significant) influence of CsA treatment on the antigenspecific IgM response in minipigs is in agreement with observations in dogs [50], cynomolgus monkeys [51] and humans with uveitis [52]. However, inhibition by CsA of the KLH-specific primary IgM response was observed in various rodent studies using different treatment schedules and routes of KLH immunization [30,48,49,53]. The KLH specific secondary IgG response in CsA treated minipigs was reduced in females and to a lesser extend also in males.…”