Development of an ELISA for nitrazepam based on a monoclonal antibody

We report the production of a specific monoclonal antibody against Nitrazepam (NZP). First, the hapten 7-aminonitrazepam (7-ANZP) was synthesized, then the hapten was coupled with bovine serum albumin and ovalbumin by the diazotization method, and used as immunogen and coating antigen. Factors affecting binding, ionic strength, pH, and methanol concentration in phosphate-buffered saline, were optimized. The monoclonal antibody had a satisfactory IC50 of 0.2 ng/mL for NZP. The cross-reactivities with other analogs were all lower than 0.1% except for 23% with 7-NZP, which suggested that the enzyme-linked immunosorbent assay method possessed high specificity. The recoveries were in the range of 84–95%, indicating that the method could be applied for the detection of NZP in urine.