Determination Of Benzodiazepine Residues In Animal Tissues By Immunoassay Methods

The aim of the study was to develope an ic-ELISA and a colloidal gold lateral flow strip assay for the determination of benzodiazepines (BZD). Nitrazepam (NZP), clonazepam (CZP) and diazepam (DZP) were choosen for the reaserchment, which were derived, as hapten. Then, they were coupled to bovine serum albumin (BSA) and ovalbumin (OVA) to synthesize immunogen and coating antigen respectively. According to the immune protocol, six New Zealand white rabbits were immunized for five times by three immunogens during four months, and the antiserum with high titres were choosen for the experiment.A polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for the determination of NZP was developed. Influence of several physicochemical parameters such as ionic strength, pH and competete time, were selected to provide a highest sensitivity on the ELISA format. The IC50 and LOD were 1.531 ng/mL and 0.081 ng/mL respectively. The linear range was between 0.17 and 13.8 ng/mL. The recoveries of NZP in urine were ranged from 62.8% to 85.6%. The coefficients of variation (CV) of the standards and the samples were≤5.67%. The specificity was evaluated by several structurally related substances, and significant cross-reactivities were founded of clonazepam, diazepam, 7-amino-nitrazepam and 7-amino-clonazepam.But for other types of sedatives such as promethazine hydrochloride, it showed good specificity, the cross-reaction rate of≤0.1%.The colloidal gold lateral flow strip assay was also designed for the detection of NZP. The colloidal gold particle of about 18.6 nm was obtained by reducing the gold chloride with sodium citrate. The optimum pH was 9 and the optimum labeling rate of polyclonal antibody was 20μg/mL. Nitrocellulose membrane M135 of Millipore, glass fiber VL78, glass fiber SB06, absorb pad and PVC board of Shanghai Biogold Company were chosen to assemble immunochromatographic lateral flow strip. The colloidal gold-labeled anti-NZP antibody was dispensed on the glass fiber VL78 with 60μL/unit. The test line and control line were separately dispensed with 0.5 mg/mL NZP-OVA and 0.2 mg/mL goat-rabbit IgG. The detection limit of the strip was 150 ng /mL for detecting the NZP standard solution.All of the parameters of the ic-ELISA developed had reached the level of foreign standards, and it could be applied into practice; The method and data provided by the colloidal gold lateral flow strip assay could offer a lead to further research into the applicability of gold labeled lateral flow strip assays for NZP and lay a foundation for developing test strip for detecting residues of other low molecule veterinary drugs in food.