| Has been booming in recent years, the aquaculture industry, however, confronts great challenges with increasing technical barriers in international trade and growing domestic drug abuse which would cause all kinds of aquatic product safety issues. “To forge iron, one must be strong.” The most thorough way is to establish an appropriate detection method and an effective surveillance to prevent and eliminated this problem. Chloramphenicol(CAP), malachite green(MG), sulfamethazine(SM2) and sulfadimethoxine(SDM) are the crucial residues in aquatic products, so that it is imperative to establish stricter screening methods. Enzyme- linked immunosorbent assay(EIA) and lateral flow immunoassay(LFIA) are convenient, quick, highly efficient ways and are suitable for the on-site detection of these chemical residues in fishery products. In this research, we synthesized and screened highly efficient antigen, developed a monoclonal antibody(m Ab) by further screening after cell fusion, and then, we developed rapid detection assays followed by the m Ab.Firstly, we synthesized different CAP antigens with mixed anhydride method and carbodiimide method in different ratios according to the functional groups, characterized the antigen with UV-vis and SDS-GAGE, immunized and screened the best immunogen, the proper detect antigen and the best mice by ELISA. An ultrasensitive m Ab against CAP, named 6F11, was developed after cell fusion and subcloning,. An indirect competitive ELISA was developed by optimizing the main impact factors. The working conditions: CAP-OVA as detect antigen, 0.01 M p H 6.5 PBS as coating dilute, p H in washing solution and antibody dilution solution was adjusted to 7.0, coloring in 15 min. Under optimal conditions, it was detected with IC50 of 0.010 ng/m L and linear range(IC20-IC80) of 0.003–0.040 ng/m L by ic-ELISA. No cross-reactivities were detectioned against the three analogues(FFC, FFA and TAP)of chloramphenicol. Recovery rate was 98.9%-106.8% with a variation coefficient less than 10% in the detection of negative milk samples by ic-ELISA method. In order to save time in the on-site detection of bulk samples, the standard curve was established in the one-step detection, and IC50 value was 0.025 ng/m L with a linear range of 0.009- 0.067 ng/m L.A gold- labelled immunoassay based on lateral flow principle was establlised for chloramphenicol detection. The working conditions were as fo llows: 4 mL 0.1M K2CO3 solution and 15 mg CAP m Ab were added in 1 m L colloidal gold solution, 0.8 mg/m L detect antigen(C AP-BSA) was coated on NC film. The detection limit was 0.3 ng/ml in milk by naked eyes after optimization and the quantitive limit of detection was 0.263 ng/m L with a linear range of 0.116-0.596 ng/m L in the standard curve established by the intensity of T line. The same results were obtained in the LFIA and ic- ELISA methods and would meet the requirements in detection of chloramphenicol residues in milk. The limit of detection was 0.2 ng/ml and sensitivity was 0.1 ng/ml of chloramphenicol residues in fishery products, which are also suitable for rapid on-site screening of chloramphenicol residues in aquatic products.A highly sensitive monoclonal antibody against Malachite Green(2E10) was prepared in the same way, and a ic-ELISA detection method was established to detect MG residues in fish. By optimizing the main influencing factors, the working conditions were determined as follows: C EO50 as detect antigen, 0.01 M p H 6.5 PBS as coating dilute solution, p H in washing solution and antibody dilution solution was adjusted to 5.0, coloring time of 15 min. Under these conditions, the IC50 value was 0.057 ng/m L with a linear range of 0.02-0.162 ng/m L by ic-ELISA method. The cross-reactivity was 90.4% to CV and noting to the other triphenylmethane compounds. Recovery rate was 98.5%-116.3% with a variation coefficient less than 10% in the detection of fish samples by ic-ELISA method.A gold- labelled immunoassay based on lateral flow principle was establlised for MG detection. The working conditions were as follows: 1 m L 30 nm colloidal gold with a concentration of 5 nmol/L, enzyme labeled secondary antibody concentration of 0.3 mg/m L, MG m Ab concentration of 0.2 mg/m L, detect antigen(CEB30) concentration of 4.5 mg/m L was coated on NC film. The IC50 values of total MG and CV residues in fish were detected to be 0.418 ng/m L and 0.532 ng/m L, respectively. No cross-reactivity was found against other analogues of MG.. The MG residue was determined to be 195 ng/g in positive fish samples with high repeatability.An efficienct antigen was determined and a highly sensitive monoclonal antibodies against sulfamethazine(SM2, named 10D6) was prepared. The IC50 value was determined to be 0.221 ng/m L with a linear range of 0.074-0.667 ng/m L by establishment of ic-ELISA method. When it was applied for recovery experiment in fish samples, the average recovery rate was 79.6%-108.6% and the variation coefficient was less than 10%.An efficienct antigen was screened and a highly sensitive monoclonal antibodies against sulfadimethoxine(SDM, named 6C11) was prepared. The IC50 value was 0.419 ng/m L with a linear range of 0.158-1.110 ng/m L by ic-ELISA method. The avera ge recovery rate was 96.3%-128.1% and the variation coefficient was less than 10%, indicating the feasibility of SDM residues detection in fishery products. |
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