黄曲霉毒素B_1人工抗原及鼠源多克隆抗血清的制备

Preparation and identification of Aflatoxin B_1 artificial antigen and its mice polyclonal antiserum

目的:研究合成并鉴定了黄曲霉毒素B1(aflatoxin B1,AFB1)人工抗原,通过动物免疫生产亲和力高、特异性好的鼠源AFB1多克隆抗血清。方法:采用N-羟琥珀酰亚胺酯(N-hydroxy-succinimide NHS)法将AFB1分别偶联于载体蛋白BSA和OVA上,分别合成免疫抗原AFB1-BSA和包被抗原AFB1-OVA,采用紫外分光光度法和SDS-PAGE进行鉴定,通过动物免疫实验获得鼠源多抗血清,并使用间接ELISA和阻断ELISA的方法对多抗血清进行了鉴定。结果:结果表明半抗原AFB1分别和载体蛋白BSA及OVA偶联成功;免疫的6只小鼠效价均达1×10-4以上,3号小鼠多抗血清敏感性最好,半数抑制浓度IC50(50% inhibitive concentration,IC50)为8.199ng/mL。结论:实验成功合成了AFB1人工抗原,并生产了高效价、高敏感性的鼠源多克隆抗体血清,为AFB1单克隆抗体制备及其快速检测试剂的研制奠定了坚实的基础。

Objective：The goal of this study was to synthesize artificial antigen of AFB1 and obtain its mice polyclonal antiserum. Method：Immunogen AFB1-BSA and coating antigen AFB1-OVA were synthesized using NHS by linking carrier proteins BSA and OVA to AFB1 and identified by ultraviolet scanning,SDS-PAGE. The mice polyclonal antiserum was obtained by BALB/C mice immunization and was identified by indirect ELISA and blocking ELISA. Result：The results showed that the hapten AFB1 was successfully linked to carrier proteins. Indirect ELISA showed a high titre above 1×10-4 of the antiserum of all the six BALB/c mice. The sensitivity of antiserum of No. 3 mice was the best,which IC50 was 8.199 ng/mL. Conclusion：Artificial antigen AFB1-BSA and AFB1-OVA were synthesized and sensitivity AFB1 polyclonal antiserum has been generated in this study,which laid a foundation base for the preparation of monoclonal antibodies and rapid test reagent against AFB1.