Furazolidone, Nitrofurantoin And Gatifloxacin Chemical Luminescence Enzyme-linked Immunosorbent Assay Method Established,

With the fast economic growth,the modern animal husbandry has developed very rapidly in recent years.And the drug residue caused by the abuse of antibiotics has become serious economic and social problems which threat human health.Nitrofurans and(Fluoro)quinolones are artificial synthesized broad-spectrum antibiotics.Because they are cheap and effective,they are widely used to treat animal diseases or as feed additives.But the abuse of these drugs result in inevitable drug residues,causing side-effects such as carcinogenicity and mutagenicity;contributing to bacterial resistance and entering food chains to cause recessive side effects.Now many countries have set MRLs(Maximum Residue Limits) for Nitrofurans and (Fluoro)quinolones.Therefore,it's necessary to establish a fast,convenient and reliable method to detect the residue of Nitrofurans and(Fluoro)quinolones to protect human health and economic benefits of the country.Many countries have established some methods to detect residue of these drugs,including traditional instrumental analytical method,microbial detection method and newly developed enzyme-linked immunoassays method(ELISA).The traditionally instrumental analytical methods include HPLC,LC-UV,LC-MS, LC-MS-MS and so on.These methods are sensitive,accurate,highly specific and can be used as standard methods.But they require expensive and heavy equipment,large volume of solvents,derivative treatment,time-consuming sample clean-up process and trained operator so they are not suitable to be used as routing screen and high-capacity field detection methods for drugs.The microbial detection method can be used to do high-capacity field detection,but because of its poor specificity,it can't be used to do qualitative and quantitative analysis.The enzyme-linked immunoassays method(ELISA) overcomes the disadvantages of the methods mentioned above.It's a rapid,sensitive,convenient method and can be used to do high-capacity field detection.ELISA method combined with chemiluminescent analysis(CL-ELISA), becomes a trend of development of ELISA because of its higher sensitivity and lower detection limit.Therefore,the aim of our current research is to establish a CL-ELISA method for the detection of furazolidone,nitrofurantoin and gatifloxacin.To develop a CL-ELISA method to detect the residue of furazolidone.We synthesized the immunogen and coating-antigen from the metabolite of furazolidone, AOZ.The immunogen was obtained via the acid anhydride scheme,AOZ was coupled with CBA and derivatived to form CPAOZ,then CPAOZ was coupled with cBSA via the acid anhydride scheme and immunogen was synthesized.Polyclonal antibodies were produced through the rabbit immunization procedure.AOZ was coupled with OVA via the glutaricdialdehyde scheme to prepare the coating-antigen. For furazolidone,the antibody showed excellent specificity and sensitivity with IC₅₀ of 0.8ng/mL,detection limit of 0.01ng/ml and no cross reaction with most related species and compounds except with CPAOZ.The result obtained by conventional colorimetric ELISA was IC₅₀ of 2.9ng/mL,detection limit of 0.2ng/ml.Compared with conventional colorimetric ELISA using the same antibody,the developed CL-immunoassay shows a significant improvement in sensitivity and detectability. The recovery rates of furazolidone spiked in feeding water samples was listed below: 88-103%for intra-assay and 78-113%for inter-assay.The CVs are in the range of 5-27%for intra-assay and 4-18%for inter-assay.The CL-ELISA method to detect the residue of nitrofurantoin was developed in the same way.The immunogen and coating-antigen were from the metabolite of nitrofurantoin,AHD.AHD was derivatived to CPAHD and immunogen was obtained via the acid anhydride scheme.The coating antigen was prepared via the glutaricdialdehyde scheme.For nitrofurantoin,the antibody showed excellent specificity and sensitivity with IC₅₀ of 0.85ng/ml,detection limit of 0.01ng/ml and no cross reaction with most related species and compounds except with CPAHD.The result obtained by conventional colorimetric ELISA was IC₅₀ of 3.2ng/mL,detection limit of 0.1ng/ml.The recovery rates of nitrofurantoin spiked in feeding water samples was listed below:93.2-110%for intra-assay and 87.6-113.5%for inter-assay. The CVs are in the range of 6-19%for intra-assay and 4-10%for inter-assay. For gatifloxacin,the immunogen and coating-antigen were obtained via the EDC scheme.The IC₅₀ of the antibody was 0.4ng/ml,detection limit was 0.01ng/ml.the antibody also showed good specificity towards gatifloxacin.The result obtained by conventional colorimetric ELISA was IC₅₀ of 2.6ng/mL,detection limit of 0.5ng/ml. The recovery rates of gatifloxacin spiked in milk samples was listed below:93-103% for intra-assay and 89-105%for inter-assay.The CVs are in the range of 5-16%for intra-assay and 4-10%for inter-assay.In conclusion,we have successfully prepared the immunogen,coating-antigen and polyclonal antibodies of furazolidone,nitrofurantoin and gatifloxacin.CL-ELISA method based on the antibodies was established.And identified CL-immunoassay shows a significant improvement in sensitivity and detectability compared with traditional ELISA method.