Enzyme linked immunosorbent assay for detecting benzodiazepines in urine

Abstract

A relatively simple ELISA technique was developed for the detection of a range of benzodiazepines (BZs) in urine. The assay employs a mouse anti-oxazepam antibody that is highly specific for the BZs. The limit of detection using 10 µl samples of urine was 0.3 µg ml^–1 oxazepam. N-Desmethyldiazepam showed equal cross-reactivity to oxazepam, 11 BZs cross-reacted weakly and flurazepam and chlordiazepoxide did not cross-react at levels reported to be found in urine. No cross-reactivity was observed with drugs of abuse and a range of therapeutic drugs commonly found in urine. The assay was used as a screen to detect the presence of BZs in urine from 88 addicts that had been screened by the EMIT technique and a radioreceptor assay (RRA) for BZs. The ELISA produced two false negatives that were EMIT and RRA positive whereas the EMIT produced four different false negatives that were positive by both ELISA and RRA. Thirty-three positives were common to all three assays. The ELISA was also used to monitor nitrazepam-like activity in the urine of a greyhound receiving 5 mg oral medication and the results were compared with those obtained by RRA. Both assays were able to detect nitrazepam-like activity for up to 10 h post-administration.