巴氯芬人工抗原的合成及其鼠源多克隆抗体的制备

Synthesis of baclofen artificial antigen and preparation of murine polyclonal antibody

【目的】合成巴氯芬(baclofen,BA)人工抗原并制备其多克隆抗体,为巴氯芬免疫学快速检测方法的建立奠定基础。【方法】采用碳二亚胺(EDC)法将BA分别与牛血清白蛋白(BSA)和鸡卵清白蛋白(OVA)偶联,合成免疫抗原BA-BSA和包被抗原BA-OVA,通过紫外扫描和SDS-PAGE凝胶电泳鉴定其偶联效果。将免疫抗原BA-BSA免疫BALB/c小鼠,利用间接ELISA、间接竞争ELISA及交叉反应试验分别对所获多克隆抗体的效价、敏感性、特异性等免疫学特性进行鉴定。【结果】紫外扫描和SDS-PAGE凝胶电泳结果表明人工抗原偶联效果良好。ELISA结果表明,多克隆抗体的效价达到1∶25600,半数抑制浓度(IC50)为22.33 ng/mL,且与巴氯芬结构类似物及其他瘦肉精类药物(γ-氨基丁酸、氯丙那林、可乐定、赛庚啶、克伦特罗、沙丁胺醇、莱克多巴胺)交叉反应率均小于0.3%,具有良好的敏感性和特异性。【结论】成功合成了巴氯芬人工抗原,并制备了免疫学特性良好的巴氯芬鼠源多克隆抗体。

【Objective】This study synthesized artificial antigen of baclofen(BA)and obtained murine polyclonal antibody with good immunological characteristics to provide an effective antibody for the research of rapid immunology detection method of baclofen.【Method】In this study,baclofen was conjugated with bovine serum albumin(BSA)and ovalbumin(OVA)by EDC method to synthesize immune antigen BA-BSA and coated antigen BA-OVA.The conjugation effect of artificial antigen was identified by UV scanning and SDS-PAGE.BLAB/c mice were immunized with immune antigen to obtain polyclonal antibody.The immunological characteristics(titer,sensitivity and specificity)of obtained polyclonal antibody were identified by indirect ELISA,indirect competitive ELISA and cross reaction test.【Result】UV scanning and SDS-PAGE results showed that the conjugation effect of artificial antigen was good.ELISA results showed that polyclonal antibody titer reached 1∶25600,and half-inhibitory concentration(IC50)was 22.33 ng/mL.The cross reactivity with structural analogues and other lean meat powder drugs(γ-aminobutyric acid,clorprenaline,clonidine,cyproheptadine,clenbuterol,salbutamol and ractopamine)were all less than 0.3%,showing good sensitivity and specificity.【Conclusion】This study successfully synthesized baclofen artificial antigens and prepared polyclonal antibody with good immunological characteristics.