Validation of a microtiter plate ELISA for screening of postmortem blood for opiates and benzodiazepines

The object of this study was to evaluate the suitability of the Neogen Corp. microtiter plate enzyme-linked immunoassays (ELISA) for opiates and benzodiazepines for screening of postmortem blood. Ninety postmortem whole blood specimens were obtained from drug-involved deaths which had been screened and confirmed positive for opiates and/or benzodiazepines. Forty negative specimens were obtained from non-opiate-involved deaths. Specimens were tested using the Neogen Opiates Group and Neogen Benzodiazepines Group microtiter plate ELISA assays. No matrix effects were found for whole blood in these assays and a dilution of 1:5 was chosen to facilitate pipetting and to bring the IC50 of the microtiter plate ELISA assay within the range of opiates and benzodiazepines encountered in medical examiner specimens. True positive, true negatives, false positives, and false negatives were determined and graphed for the ELISA results against gas chromatography-mass spectrometry (GC-MS), gas chromatography-nitrogen-phosphorus detection and case histories. From these graphs and the ROC curves, the optimal cut-off for the Neogen Opiates Group ELISA was found to be between 20 and 50 ng/mL morphine equivalents and the optimum cut-off for the Neogen Benzodiazepines Group ELISA was between 20 and 50 ng/mL temazepam equivalents. The Neogen Opiates Group ELISA had a sensitivity of 95.2% +/- 2.7% and a specificity of 92.2% +/- 3.4% versus GC-MS at a cut-off of 20-ng/mL cut-off and a sensitivity of 88.8% +/- 3.9% and specificity of 96.8% +/- 2.1% versus GC-MS at a 50-ng/mL morphine equivalents cut-off. The Neogen Benzodizepines Group ELISA had a sensitivity of 100% +/- 1.3% and a specificity of 94.6% +/- 2.9% versus GC-MS (20-ng/mL temazepam equivalents cut-off) and a sensitivity of 95.8% +/- 2.5% and specificity of 98.2% +/- 1.8% versus GC-MS at a 50-ng/mL cut-off.