| In recent years, the problem of drug residues in aquatic products become more and more serious, which impact on China's aquatic products export trade and harm human health. Along with the increase of regulation, high sensitive, high-throughput, quick and easy become a trend in future technique. In this research, three kinds of important risk factors including quinolones antibiotics, quinoxaline growth promoters and methylene blue disinfectant were selected as research object, with the purpose of deveploment of rapid detection assays by antigen design and antibody preparation.This research designed and synthezied ten kinds of hapten and corresponding antigen. Best immununogen were obtained by mice immunization and antiserum screening. Three kinds of antibody with different regonization to quinolones antibiotics were obtained after cell fushion and antibody preparation. The sensitivity of method was greatly improved by heterogenous coating. The antibody 1C9, with NOR-NH2-KLH as immunogen and PAZ-GA-OVA as coating, IC50 of the developed ELISA for NOR was 0.12 ng/m L, linear range was 0.05-0.25ng/mL, cross-reactivity(CR) experiments showed good CR with 20 quinolone antibiotics. The antibody 1E5, with NAD-NH2-KLH as immunogen and PAZ-GA-OVA as coating, IC50 of the developed ELISA for NAD was 0.19 ng/m L, linear range was 0.05-0.74ng/m L, CR experiments showed good CR with 14 quinolone antibiotics. The antibody 3E4, with mixture of SAR-COOH-KLH and NOR-COOH-KLH as immunogen and SAR-OVA as coating, IC50 of the developed ELISA for SAR was 0.52ng/mL, linear range was 0.09-2.94ng/mL, CR experiments showed good CR with 21 quinolone antibiotics.An immunochromatographic strip based on antibody 3E4 that recognizes 32 quinolone antibiotics was prepared. The optimal working condition were as follow: 6?L 0.1 mol/L K2CO3 and 40?L 0.2 mg/mL were added in 1 m L colloidal gold solution. The cut-off values of the strip range from 1 to 100 ng/m L and the limits of detection are 0.1–10 ng/mL. this immunochromatographic strip is a very useful tool for the primary screening of quinolones in milk.A broad specific mAb against quinoxaline growth promoters were successfully prepared. Furthermore, using the broad specific mAb 2F3, an indirect competitive ELISA for quinoxaline growth promoters detection in fish feed was developed using MQCA derivative MQCA-ACA coupled to OVA as the heterologous coating antigen. Under optimized assay conditions( the concentration of coating and antibody were 0.3 ?g/m Land 0.11 ?g/mL, 0.01 M pH 8.2 BB with 1.6% of NaCl as standard dilute), the IC50 values was 1.03ng/ml for OLA, the cross-reactivity with MEQ, QCTand CBX were 67%,60% and 0.9%. The recoveries ranged from 82.1% to 96.3%, the developed icELISA was rapid and reliable for the determination of sum of OLA, MEQ, and QCT in fish feed.A high specific mAb against MQCA was successfully prepared by design and sythesized five MQCA haptens with exposuring different antigenic determinant. Using this mAb 1A11 prepared from mouse immunized by immunogen MQCA-(NH2)4-KLH, an indirect competitive ELISA for MQCA detection in fish was developed using MQCA derivative MQCA-NH2 coupled to OVA as the heterologous coating antigen. Under optimized assay conditions( the concentration of coating and antibody were 0.03 ?g/mLand 0.3 ?g/mL, 0.01 M pH 7.4 PBS with 1.6% of NaCl as standard dilute), the IC50 values was 3.17 ng/mL for MQCA, linear range was 0.58-17.29 ng/m L The recoveries ranged from 81.6% to 90.7%, the developed icELISA was rapid and reliable for the determination of MQCA in fish.An immunochromatographic strip for MQCA based on antibody 1A11 was prepared. The optimal working condition were as follow: 4 ?L 0.1 mol/L K2CO3 and 50 ?L 0.2 mg/mL were added in 1 mL colloidal gold solution. The cut-off values of the strip range 5 ng/m L and the limits of detection are 0.25 ng/m L. This immunochromatographic strip is a very useful tool for the primary screening of MQCA in fish.A high specific mAb against methylene blue(MB) was successfully prepared by design and synthesis of a new hapten MB-HS. Using this specific mAb1H4, an indirect competitive ELISA for MB detection in fish was developed. Under optimized assay conditions(the concentration of coating and antibody were 0.1?g/mLand 0.1?g/m L, 0.01 M pH7.4 PBS with 1.6% of NaCl as standard dilute), the IC50 values was 21.13 ng/m L ng/ml for MB, linear range was 4.26-104.83 ng/mL The recoveries ranged from 82.1% to 90.1%, the developed icELISA was rapid and reliable for the determination of MB in fish. |
PDF Full Text Request