Development and Evaluation of a Microtiter Plate Enzyme Immunoassay for Antibodies to 3,3'-Dichlorobenzidine

The authors obtained and evaluated antisera from rabbits injected with a derivative of a potent bladder carcinogen, dichlorobenzidine (DCB), conjugated to bovine serum albumin (BSA). A 14C-radioimmunoassay (RIA) was able to detect the presence of DCB antibodies, but its relative insensitivity led to the development of a more sensitive enzyme Immunoassay (EIA). The EIA test was a "sandwich" method in which a second antibody, labeled with an enzyme (horseradlsh perox|dase), was used to measure antlbody bindlng to transferrin (Tf)-conjugated DCB immobllized on a microtiter plate. Antibody titers measured by RIA were approximately 1:40; when measured by EIA, they were approximately 1:40,000. Antibody specifictty was assessed by comparing the antibody binding actlvlties of DCB, BSA, Tf, BSA-conjugated to DCB, and a number of N-substltuted aromatlc compounds that tncluded benzldlne (Bz). Among the compounds tested, the rabbit antiserum reacted only with DCB and the carrier protein, BSA. Moreover, antibody binding activity to Tf.conjugated DCB was significantly inhlbited by unconjugated DCB concentrations between 30 and 500 ng/mL. The precision of antibody blnding activities as a functlon of DCB concentration (expressed by the CV) ranged from 9% for Iow (30 ng/mL) DCB levels to 12% for higher (500 nglmL) levels. This evaluation suggests that the antlserum obtained would be appropriate for detecting DCB levels at the ng/mL level.