An enzyme-linked immunosorbent assay for measuring anti-sheep red blood cells antibodies in lead-exposed toads

Abstract

Introduction: Immune function assays to screen immunotoxic effects of xenobiotics has recently become of major interest. In the framework of our studies, we standardized methods to quantify the humoral response of an amphibian species ( Bufo arenarum, Amphibia, Anura) exposed to sublethal lead (as acetate). Methods: The levels of agglutinins to heterologous red blood cells (RBC) were measured in serum from adult B. arenarum. Since agglutinin titers were very low, a noncompetitive enzyme-linked immunosorbent assay (ELISA) method was carried out. As toad serum showed marked nonspecific binding, we developed a new ELISA on microtiter plates for the quantitative determination of the heterophile antibodies. The method was based on that described by Hirvonen et al. [Vox Sang. 69 (1995) 341], employing sheep red blood cells (SRBC) sensitized with amphibian antibodies that were transferred to microplates; later the measurement of bound immunoglobulins was performed. Different variables such as the amount of antigen, blocking agents, and other experimental conditions (fixing solution and commercial plates) were studied. Toads ( n=22) received a weekly subcutaneous injection of 50 mg/kg lead (acetate) for 6 weeks, and the control ones ( n=26) were injected with Na acetate at the same time. Results: The anti-sheep RBC antibodies titers of adult toads were obtained with the improved ELISA method, being the absorbance range 0.12 to 1.58 AU (1/200 diluted serum). Titers from lead-exposed toads were also determined, being the final titers (expressed as x̄±S.E.M.) higher (0.79±0.06 AU), than those of Day 0 (0.57±0.06) ( P<.01). Discussion: It was concluded that the ELISA technique we developed was useful for measuring the humoral immune response in this animal model and that in these preliminary studies, lead showed an immunostimulating action on the humoral immune system.