Abstract
The production of antigen-specific antibodies represents a major defense mechanism of humoral immune responses. Several assays have been developed to assess T-cell-dependent antibody responses (TDAR). Of these assays, the antibody forming cell assay (AFC) or plaque forming cell (PFC) assay and ELISA are the two most often used tests to assess immunotoxicity. Historically, the T-cell-dependent antigen of choice has been sheep red blood cells (SRBC). The SRBC AFC assay is considered the “gold standard” for TDAR based on extensive intra- and inter-laboratory validation in mice and has been utilized for over 35 years. The quantification of the primary AFC response (i.e., the specific IgM antibody-forming cell response) was found to provide one of the best predictors of immunotoxicity in mice. The SRBC-specific ELISA is relatively new, with the first publication of the method appearing in 1993. Data from the application of using both the SRBC specific AFC and ELISA for evaluation of potential immunotoxicity of chemicals in rodents and the pros and cons and associated issues of each method were presented. Specifically, the following was discussed: (1) studies investigating the incorporation of the SRBC-specific IgM ELISA in rats on standard toxicology study; (2) characterization of an approach to developmental immunotoxicology assessment in the rat using SRBC as the antigen; and, (3) data from an inter-laboratory study comparing the AFC assay and ELISA in outbred rodents using both cyclophosphamide and dexamethasone.
INTRODUCTION
The production of antigen-specific antibodies represents a major defense mechanism of humoral immune responses. Following antigen exposure, the generation of an antigen-specific antibody response involves the cooperation and interaction of several immune cell types (i.e., antigen-presenting cells, T-helper and B-cells) and cell products (e.g., cytokines). Several days after antigen exposure, antigen-specific antibodies, predominantly of the IgM isotype, are generated and released into the general circulation by B-cells and plasma cells (terminally differentiated B-cells). There are, thus, numerous targets that may be altered following xenobiotic exposure, making assays that evaluate antigen specific antibody production a relatively comprehensive and sensitive assessment of immune function.
Data indicate that the primary antibody response to SRBC may be one of the most sensitive endpoints available to assess chemical-induced alterations to the murine immune system (Luster et al., Citation1988, Citation1992). As the assessment of the primary antibody response to a T-dependent antigen (e.g., SRBC) has been reported to provide one of the best predictors of immunotoxicity in mice (Luster et al., Citation1992), this endpoint has subsequently become the cornerstone of recent guidelines for assessing the potential immunotoxicity of xenobiotics. Although a number of assays have been developed to evaluate antibody production, the antibody forming cell (AFC) assay or plaque forming cell assay (PFC) and enzyme-linked immunosorbent assay (ELISA) are the two most frequently employed to evaluate the potential immunotoxicity of a xenobiotic. For example, the United States Environmental Protection Agency (EPA) in 1998 published, and subsequently finalized in 2005, guidelines (USEPA Health Effects Test Guidelines. OPPTS 870.7800. Immunotoxicity) requiring chemicals used as pesticides to initially undergo an evaluation of the primary humoral immune response to a T-dependent antigen (i.e., SRBC) utilizing either the AFC assay or ELISA following test article exposure for 28 days.
Antibody Forming Cell Assay
The AFC involves the removal of spleens from animals that have been immunized with SRBC and the subsequent in vitro production of IgM antibody specific for SRBC, which in the presence of complement produce plaques (i.e., clear areas of hemolysis around each antibody forming cell). The plaques are then visually quantitated and the resulting data expressed as SRBC-specific IgM AFC (or PFC)/spleen or IgM AFC (or PFC)/million spleen cells. Data are expressed as both AFC/spleen and AFC/million cells in order to help elucidate the potential contribution of xenobiotic-induced alterations of spleen cell numbers on the response. For example, a particular xenobiotic may produce a general decrease in the various cell types in the spleen, which may result in the PFC/spleen being decreased and the PFC/million cells unaltered. Such data would suggest that general humoral immunocompetence may be altered, while the specific activity of humoral immunity is not. Following a second immunization with SRBC, the secondary IgG AFC immune response can then be assessed using a slight modification of this assay (Holsapple, Citation1995).
The AFC assay has been well-characterized across multiple labs, and is likely the most validated endpoint in immunotoxicology. The AFC assay can also be conducted entirely in vitro using immunocompetent cells obtained from either treated or naive animals. This approach allows for studies to evaluate mechanisms of action of xenobiotic-induced immunotoxicity (i.e., separation-reconstitution studies; xenobiotic-induced direct vs. indirect effects) (Holsapple, Citation1995). The AFC assay, however, does not quantitate the amount of antibody produced, but rather the number of specific antibody-producing plasma cells in a particular tissue (i.e., the spleen), and thus, does not account for antibody produced in other sites (e.g., bone marrow, lymph nodes). Therefore, it is difficult to extrapolate results of the AFC assay to human tests since serum antibody concentrations are typically measured in humans. In addition, the AFC assay requires the sacrifice of the animal and the harvested spleen cells must be “plaqued” within 18–24 hours after collection and therefore limits experimental design relative to exposure. The AFC assay is also very labor intensive and not conducive to automation ().
TABLE 1 Antibody forming cell assay
SRBC-Specific IgM ELISA
The SRBC-specific IgM ELISA utilizes solubilized, hemoglobin-free SRBC membranes and measures SRBC specific antibody found in the serum generated from all antibody-producing tissues (i.e., spleen, lymph nodes, bone marrow), reflecting the systemic humoral immune response (Temple et al., Citation1993). No commercial SRBC membrane preparations are available; each laboratory must make their own. Further, no standard is currently available for the assay and therefore, it is important that appropriate assay criteria be established and utilized. For each serum sample, it is recommended that a semi-log graph be initially created and the linear portion of the curve identified by using a log-log curve fit. At least one point above and at least one point below 0.5 OD should then be selected, using a minimum of three points, and a slope not less than −0.600 but not exceeding −1.200 (a slope of −1.000 is optimal) obtained. The linear region of the curve is then interpolated at 0.5 OD. The serum titer obtained is usually reported as log2 titer. Alternatively, endpoint titers may also be used rather than the method described above. Endpoint titers are easier to measure and they do not depend on the generating a linear region on the dilution curve (difficult to obtain for low titer samples).
Compared to the AFC assay, the ELISA can be automated, as it is typically performed in 96-well plates. Furthermore, the ELISA allows for multiple serum samples to be taken from the same animal and stored frozen for later analysis. A timecourse of humoral immune function, therefore, can be conducted, a recovery period following test article administration may be evaluated or, upon antigen re-challenge, the secondary immune response measured. Unlike the AFC assay, however, the ELISA is not conducive for mechanistic investigations (e.g., separation-reconstitution studies). In addition, standard analysis criteria (e.g., log-log slope values) for assay acceptance need to be established. Further, there is no commercial source for a critical assay reagent (i.e., the SRBC membrane preparation), with each laboratory making their own preparation (). Data on the incorporation of the SRBC-specific ELISA for hazard identification purposes in rats in a standard toxicology study was also discussed. Rats dosed for 30 or 90 days with either cyclophosphamide or carbon tetrachloride and immunized with SRBC 6 days prior to sacrifice were subjected to standard toxicology endpoint (i.e., organ weights, clinical chemistry, hematology, and histopathology) analysis (Ladics et al., Citation1995, Citation1998). Results suggest that the SRBC-specific ELISA can be utilized to assess humoral immune function for hazard-identification purposes in rats in a standard toxicology study.
TABLE 2 SRBC-specific ELISA
Developmental Immunotoxicology
Immunotoxicology procedures, such as the primary humoral immune response to SRBC, have been developed for assessing xenobiotic-induced alterations of the immune system of adult rodents (Luster et al., Citation1992). Increasing attention, however, has been focused on the effects of xenobiotics on the developing immune system (Holladay and Smialowicz, Citation2000; Dietert et al., 2002; Holsapple et al., Citation2005). Data on whether the SRBC primary antibody response would be useful in assessing the functionally immature immune system of young animals (e.g., neonates) was discussed (Ladics et al., Citation2000). Ten-day-old rat pups were unable to generate a primary IgM response to SRBC using either the AFC assay or ELISA due to the apparent immature status of their immune system. Results with weanlings suggest that an antibody response to SRBC of sufficient magnitude can be demonstrated with the AFC assay, however, a high background response was observed with weanlings when using the SRBC-specific IgM ELISA that may prevent its use with these age animals.
Interlaboratory Comparison of the SRBC Primary Antibody Response
Results from a two-phase inter-laboratory study comparing the sensitivity of the AFC vs. ELISA in detecting immunosuppression in outbred rodents were also presented. A catalyst for this study involved answering the following question “Is it possible that a chemical could affect the number of splenic AFCs without affecting the amount of secreted antigen specific IgM antibody in the sera of rodents”? The study involved four immunotoxicology laboratories evaluating the primary antibody response in female CD rats and CD1 mice to cyclophosphamide (CY) in phase I and dexamethasone (DEX) in phase 2. All participants used the same source of SRBC to establish optimal concentrations for immunization and peak day of the antibody response, but assay protocols specific to each lab. Compared to inbred rodent strains, the antibody response in outbred strains tends to be more variable.
The order of sensitivity for CY was as follows: rat AFC > mouse AFC > rat ELISA > mouse ELISA. There was a greater inhibition of the antibody response observed for the AFC vs. ELISA in both species. However, the CP dose at which statistical significance was first observed in both species was quite similar within each lab using either assay (Loveless et al., Citation2007; ). For DEX the order of sensitivity was identical to CY. The AFC assay detected significant and greater suppression at lower concentrations of DEX compared to the ELISA in both species. All labs detected DEX suppression using the rat AFC vs. only one lab using mouse ELISA (Loveless et al., Citation2007; ). For both compounds the magnitude of suppression was greater using the AFC assay. These data indicate that the AFC assay is more capable of consistently identifying suppression of a T dependent antibody response across laboratories in outbred rodents. Additional compounds, however, should be evaluated before concluding that one method is better or more sensitive to the other in detecting suppression of the antibody response.
TABLE 3 Lowest dose of cyclophosphamide at which statistical significance was observed
TABLE 4 Lowest dose of dexamethasone at which statistical significance was observed N.S.- not significant
SUMMARY
The TDAR has become an integral part of immunotoxicology testing. The AFC or ELISA utilizing the T-dependent antigen SRBC has historically been conducted to assess the primary humoral immune response. With both assays, however, variability in the antibody response has been reported to occur when outbred rodents are utilized. Data suggest that the SRBC-speciic ELISA may be incorporated into standard toxicology studies (Ladics et al., Citation1995; Citation1998), but its use for DIT may be limited (Ladics et al., Citation2000). In contrast, the AFC assay has been reported to be useful for DIT studies involving post-weaning animals (Ladics et al., Citation2000). Data indicate that the AFC assay is better at identifying suppression of a T-dependent antibody response across laboratories in outbred rodents (Loveless et al., Citation2007). However, further evaluation of additional compounds should be conducted before concluding that one method is superior or more sensitive to the other in detecting suppression of the antibody response.
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