Preparation Antibody And Establishment ELISA Method Of1,4-bisdesoxycyadox And1,4-bisdesoxymequindox

Cyadox and mequindox are synthetic veterinary drugs, which belong to the quinoxaline drugs, have a good antibacterial and growth promoting effect and are widely used in fish breeding and poultry rasing. Researches showed that the toxicity of cyadox is low when compared with other veterinary drugs, and is with good security. But it had been reported that some metabolites of cyadox have potential toxicity to animals and could cause food safety problems. The toxicity of mequindox is higher, long-term use or abuse can endanger the health of animal and human. Therefore, to establish a rapid, sensitive detection method to monitor cyadox and mequindox residues in edible animal tissue is significant. In this paper,1,4-bisdesoxycyadox and1,4-bisdesoxymequindox are choosed as the research objects and the aim is to establish a rapid, sensitive, high throughput and antibody-based enzyme-linked immunosorbent assay (ELISA).1,4-bisdesoxycyadox (Cy4) is the main metabolite of cyadox and has been identified as the marker residue. According to the structure of Cy4, carboxymethyl hydroxylamine oximation was used to introduce a carboxyl, then, the hapten Cy4coupled with carrier protein (BSA and OVA) through the mixed anhydride method. Cy4-BSA was the immune antigen and Cy4-OVA was the detection antigen. Polyclonal antibodies with high titers and sensitivity were obtained after immunized female Balb/C mice. Three hybridoma cell lines (A3C5, D9D2and F10D2) were obtained by the fusion of spleen cells and mouse myeloma cells Sp2/0through screening and cloning. Monoclonal antibodies were prepared and purified by caprylic acid ammonium sulphate method. The subclass and light chain of the three monoclonal antibodies were all IgG2b and λ light chain. The titers were1:128000-1:512000. No cross reaction with other structural analogues was detected and the monoclonal antibodies had good specificity.Monoclonal antibody from D9D2hybridoma cell line was chosed to establishment the ELISA method. By optimizing the reaction conditions, the standard curve of the indirect competitive ELISA (ic-ELISA) was established. The sensitivity (IC50) was50ng/mL, the sensitivity was200times higher than that of the polyclonal antibody, the linear range (IC20-IC80) was6.2to481.1ng/mL. Fortified with concentrations4-4000μg/kg of Cy4to the chicken muscle, the recovery rate was71.36%-83.10%, and the intra-assay and inter-assay precisions were both<10%. This study established a monoclonal antibody-based enzyme-linked immunosorbent method with high sensitivity and good specificity, which can be used to detect the cyadox residues in animal edible tissues.1,4-bisdesoxymequindox (M4) is the main metabolite of mequindox and has been identified as the marker residue. According to the structure of M4, carboxymethyl hydroxylamine oximation was used to introduce a carboxyl, then, the hapten M4coupled with carrier protein (BSA and OVA) through the mixed anhydride method. M4-BSA was the immune antigen and M4-OVA was the detection antigen. Polyclonal antibodies with high titers were obtained after immunized female Balb/C mice. The titers were1:128000-1:512000; the sensitivity (IC50) were0.32μg/mL-12.38μg/mL; the antibodies only react with few of its structural analogues and the antibodies had good specificity. The polyclonal antibody with the best sensitivity was chosed to establishment the ELISA method. Fortified with50-5000μg/kg of M4to the swine liver, the recovery rate was73.65%-85.82%, and the intra-assay and inter-assay precisions were both<15%. This study established a polyclonal antibody-based enzyme-linked immunosorbent method with high sensitivity and good specificity, which can be used to detect the mequindox residues in animal edible tissues.