Preparation Of Immunomagnetic Beads And Imunoaffinity Chromatographic Column For The Metabolite Of Furazolidone(AOZ)

Veterinary drugs play an important role in modern farming.But in recent years,some farmers are in order to save costing and acquire a good antibacterial effect,they misapplication or illegal use of veterinary drugs,that result in the residues of veterinary drugs in animal-derived food and harmfulness to the health of human beings and natural environment.Nitrofurans belongs to a broad spectrum antibiotic,for the majority of Gram-positive bacteria and Gram-negative bacteria have antibacterial action.But because of their toxicity of carcinogenic,mutagenic and teratogenic,nitrofurans have been banned from using in animals as veterinary drugs in many countries.Furazolidone is one of the nitrofurans,and its metabolite is 3-amino-2-oxazolidinone(AOZ),AOZ has strong toxicity of carcinogenic and mutagenic.The method used for determination the residues of furazolidone at home and abroad include instrumental analysis and immunoassay.In order to improve the sensitivity,reduce the matrix interference and sample pretreatment time in experiment,we planned to prepare the monoclonal antibody(McAb)against AOZ,and coupled with magnetic beads and sepharose to make into the immunomagnetic beads of AOZ(AOZ-IMB)and the immunoaffinity chromatographic column of AOZ(AOZ-IAC),then optimized the conditions of test.The AOZ-IMB and AOZ-IAC were used in sample pretreatment,and evaluated by indirect competitive ELISA method.Test I Preparing and identifing the monoclonal antibody against AOZCPAOZ was conjugated with bovine serum albumin by active ester method with N-Hydroxysuccinimide.We picked up one murine with the best titer of anti-AOZ serum after fourth immunized with AOZ-BSA.The myeloma cell of murine and the spleen cell of immunized murine were fused by hybridoma technology.Inoculated positive hybridoma to abdomen of murine,and then collected ascites.The titer,specificity and sensitivity of McAb against AOZ were characterized by indirect ELISA and indirect competitive ELISA method.The titer of McAb against AOZ was 1:106.The half inhibitory concentration of NPAOZ was 0.31 ng/mL.The cross reaction studies showed that there was no cross reactivity of McAb against AOZ with NPAMOZ,NPAHD,NPSEM,p-nitrobenzaldehyde and p-formylbenzoic acid except NPAOZ.The results showed that we acquired McAb against AOZ with highly antibody titer,sensitivity and specificity.Testn ? Preparation of immunomagnetic beads for AOZAnti-AOZ McAb was coupled to magnetic beads which activated by N-Hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.Immunomagnetic beads were prepared by coupling 50 ?g of anti-AOZ McAb with 1 mg of magnetic beads.The optimized results of adsorption and desorption conditions showed that,there was a highly adsorption rates with NPAOZ when the pH of sample solution was 7.0 and the sample solution was adsorbed for 10 minutes.The NPAOZ could be eluted easily from AOZ-IMB by eluting with 300 ?L of methanol for 5 minutes.The AOZ-IMB could only specifically adsorb the NPAOZ and it could not adsorb the NPAMOZ,NPAHD or NPSEM.The adsorption rates of NPAOZ was 86.5%when the AOZ-IMB was re-used f-or twice.The recovery rates of AOZ at spiked levels between 0.25 ng/g and 2 ng/g were more than 78%,and the coefficient of variation was between 5.3%and 10.9%.The limit of detection was 0.049 ?g/kg,and it was lower than the limit of detection with common sample pretreatment which was 0.139 ?g/kg.The results indicate that the immune-extraction method based on immunomagnetic beads with anti-AOZ McAb could enrich NPAOZ in chicken effectively,and that improved the sensitivity of ELISA detection method.Test ? Preparation of immunoaffinity chromatographic column for AOZAOZ-IAC was prepared by coupling anti-AOZ McAb with sepharose-4B which activated by CNBr.Optimized the adsorption and desorption conditions,confirmed the column capacity and the reusability of AOZ-IAC,and then AOZ-IAC was used in sample pretreatment to enrich and purify the NPAOZ.Calculated the recovery rates of AOZ and sensitivity by indirect competitive ELISA method.The results showed that,the adsorption rates of NPAOZ was more than 90%when the pH of sample solution was 7.0 and the sample solution was flowed through the AOZ-IAC at the speed of 2 mL/min.The NPAOZ could be eluted easily from AOZ-IAC by eluting with 6 mL of 90%methanol-water solution at the speed of 3 mL/min,and the elution rates of NPAOZ was more than 90%.The column capacity of AOZ-IAC was 1.6?g/mL,and the AOZ-IAC could be re-used at least five times.The recovery rates of AOZ at spiked levels between 1 ng/g and 8 ng/g were more than 80%,and the coefficient of variation was lower than 10%.The limit of detection was 0.055 ?g/kg.