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Sulfaquinoxaline (Sulfaquinoxaline, SQ) is a highly effective anticoccidial and antibacterial agents, primarily for the treatment of poultry ruminant coccidiosis young animals and small animals. Farms for the prevention and treatment of animal diseases often added to animal feed or drinking water sulfaquinoxaline often leads to acute and chronic poisoning of the livestock and livestock products, drug residues, often low-dose ingestion containing sulfaquinoxaline residues food of animal origin, when up to a certain concentration in the body enrichment, can cause allergic reactions, allergic reactions, even teratogenic and carcinogenic effects. Therefore, all countries place more emphasis on the detection of residual issues, China and the European Union, Japan, the United States and other Western countries provides sulfaquinoxaline residue limits 100ng / g. In the field of drug residue detection, immunological analysis because of its simple, rapid, high sensitivity, specificity, low cost, batch testing advantage has become the focus. The purpose of this study was to establish sulfaquinoxaline residual enzyme-linked immunoassay (ELISA) method, in order to facilitate the farmers self-test, and provide a better basis for the the effective regulatory sulfaquinoxaline use and residual. Basis of the study of the molecular structure of the sulfa quinoxaline morpholine carrier protein (BSA, OVA), diazotization method of synthetic immunogen original BSA-SQ, and thus to prepare monoclonal antibodies final sulfaquinoxaline successfully developed a rapid direct competitive ELISA detection kit. The main findings are as follows: sulfaquinoxaline complete antigen preparation and identification with glutaraldehyde (GA) carbodiimide (EDC) Act, diazotization method to explore the Preparation of artificial antigen BSA-SQ, and the ultraviolet scanning (UV), SDS-PAGE electrophoresis preliminary identification. The results show that: GA law the was with EDC legal system, BSA-SQ coupling effect is not ideal, BSA-SQ is better prepared by diazotization legal system. BSA-SQ diazotization prepared by artificial antigen to immunize BALB / C mice, to obtain a high price-sensitive anti-sulfaquinoxaline polyclonal antibody (pAb) to determine the diazotization prepared artificial antigen BSA-SQ succeed . Sulfaquinoxaline of monoclonal antibodies and immunological characteristics identified by the BSA-SQ immune multi-resistant BABL / C mice, alternate mouse cell fusion experiment screened by enzyme-linked immunosorbent assay (ELISA) only; ultra after immunization, and their spleen cells with NS0 tumor cells using PEG4000 for cell fusion, HAT medium screening culture; cloned by limiting dilution method is carried out three times to obtain a capacity of 3 Sulfaquinoxaline monoclonal antibodies (mAbs) hybridoma cell line 1H8-B11-C6 1H8 1H8-F5 to; purified using the mass production of vivo inducible ascites method. Identified 1H8-B11-C6 1H8 1H8-F5 cell culture supernatant indirect ELISA titer were 1:1.92 × 10 ~ 2,1:1.92 × 10 ~ 2,1:3.84 × 10 ~ 2, ascites efficiency price 1:2.0 × 10 ~ 5,1:2.0 × 10 ~ 5,1:2.56 × 10 ~ 5, affinity assay showed that cell lines 1H8-F5 highest affinity constant (Ka) of 4.19 × 10 ~ 9 L The / moL, the blocking ELISA measured SQ IC50 13.1ng/mL, cross with the sulfa chloropyrazine Sodium response (CR) rate of 20%, and other similar compounds CR. Sulfaquinoxaline enzyme-linked immunosorbent rapid method for detection of in SQ mAb based on, prepared in accordance with direct competitive ELISA experiment principle, assembly SQ residue rapid detection of direct competitive ELISA kit (SQ-Kit). SQ-Kit standard curve regression equation y =-0.262x 0.6778, correlation coefficient R ~~ 2 = 0.9786, the SQ IC50 for 7.5ng/mL, detection limit 5.0ng/mL. The linear detection range the 5.0 ~ 128.0ng/mL, and with good specificity, reproducibility and stability. The SQ-Kit for the detection quinoxaline residues of sulfonamides in milk and meat samples work can be completed in 1.5 ~ 2h, with fast, simple, sensitive, and specific characteristics suitable for SQ residues rapid detection of large-scale qualitative and quantitative identification.
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