Validation of a radioimmunoassay procedure for N,N'-diacetylbenzidine, a metabolite of the chemical carcinogen benzidine, in urine.

The biaryl amine benzidine has been reported to produce tumors of the bladder in both man and animals. We have developed and validated a radioimmunoassay (RIA) procedure for monitoring potential human exposure to benzidine through detection in urine samples of one of its known urinary metabolites. The antibody used in the assay was produced in rabbits and is specific for N,N'-diacetylbenzidine (N,N'-DAB). At a 1/15000 initial dilution, this antiserum bound 50% of an iodinated tracer [125I]-N4'-[4-hydroxyphenethylaminohemisuccinyl]-4-acetamido-4-aminobiphenyl. One hundred pg (0.373 pmol) of N,N'-DAB displaced 50% of the tracer initially bound to the antiserum. The validity of the RIA procedure was established by the parallelism exhibited between the standard curve and aliquots of spiked human urine samples. The RIA has good spiked human urine samples. The RIA has good sensitivity (average minimal detectable dose less than 10 pg) and reproducibility (within-and between-assay coefficients of variation at midrange of the standard curve were 3.94 and 12.48, respectively). A series of 109 randomly selected human urine control samples were analyzed and a nonspecific interference of 0.52 +/- 0.04 ng of N,N'-DAB/mL urine was found.