呋喃它酮代谢物5-甲基吗啉-3-氨基-2-恶唑烷酮单克隆抗体的制备、鉴定与胶体金免疫层析试纸条的研制

目的:制备5-甲基吗啉-3-氨基-2-恶唑烷酮（AMOZ）单克隆抗体,并建立AMOZ胶体金试纸条检测方法。方法:AMOZ与对醛基苯甲酸反应生成半抗原CP-AMOZ,将CP-AMOZ与BSA、OVA偶联制备完全抗原。免疫小鼠后制备腹水型抗体,ELISA检测抗体效价与其对CP-AMOZ半抑制浓度（IC50）和最低检测限（LOD）,并检测抗体的特异性。胶体金与单抗偶联,同时在NC膜上包被完全抗原、兔抗鼠多抗分别作为检测线和质控线,制备免疫层析试纸条,测定试纸条的灵敏度与特异性。结果:成功制备CP-AMOZ-BSA、CP-AMOZ-OVA完全抗原及抗CP-AMOZ的单抗,抗体效价为1∶1.6×105,对CP-AMOZ IC50为0.86μg/L,LOD0.07μg/L,与其他硝基呋喃类药物代谢物及其衍生物均无交叉反应。制备的胶体金试纸条灵敏度5μg/L。结论:成功合成了CP-AMOZ的完全抗原,免疫小鼠后制备了特异性强的单抗,并以此为基础研制出敏感、特异的胶体金快速检测试纸条。 

Production and characterization of monoclonal antibody against the metabolite of furaltadone,5-morpholino-3-amino-2-oxazolidone (AMOZ), and establishment of colloidal gold immunochromatography for detecting

Object: The monoclonal antibody against 5-morpholino-3-amino-2-oxazolidone ( AMOZ) was prepared, and the corresponding colloidal gold immunochromatography method was established. Method: The hapten CP-AMOZ was synthesized by the reaction between AMOZ and 4-CBA and conjugated with BSA and OVA using activated ester method.Ascitic type antibody was prepared by hybridom cell lines which secret monoclonal antibody against CP-AMOZ by mean of immunizing BALB/c mice, cell fusion, ELISA screening and subcloning. Data including the titer of antibody, its IC 50 against CP-AMOZ and LOD were detected by ELISA analysis and determine its specificity. Competitive colloidal gold immunochromatographic dipstick was produced on this base, gold nanoparticles were coupled with monoclonal antibodies, CP-AMOZ-BSA and rabbit-anti-mouse polyclonal antibody were separately coated on nitrocellulose membrane as test line and control line to detect the limit of detection and specificity.Result: The titer of the mAbs was 1 ∶1.6 × 105 , the IC 50 value and LOD to CP-AMOZ were 0.86μg /L and 0.07μg/L respectively. The mAbs had little cross reactivity with other nitrofurans metabolites and derivates, showing high specificity to CP-AMOZ, and LOD of colloidal gold immunochromatographic dipstick was 5μg /L.Conclusion: The antigen of CP-AMOZ was successfully synthetized and the monoclonal antibody specific to CP-AMOZ was prepared, which help prepare the colloidal gold rapid detection dipstick of high sensitivity and specificity.