百草枯完全抗原及多克隆抗体的制备

PREPARATION OF PARAQUAT COMPLETE ANTIGEN AND ANTI-PARAQUAT POLYCLONAL ANTIBODY

目的改造百草枯(Paraquat PQ)半抗原,制备百草枯完全抗原及多克隆抗体,为建立酶联免疫吸附方法及其在食品安全快速检测中的应用奠定基础。方法改造半抗原PQ-hap,混合酸酐法合成免疫原(PQ-BSA)和包被原(PQ-OVA),采用SDS-PAGE电泳和质谱对其鉴定,免疫家兔2只,制备多克隆抗体;间接ELISA对其进行效价、灵敏度和特异性检测。结果成功合成百草枯免疫原和包被原,6次免疫后2只家兔血清的效价分别达到1∶40 000和1∶160 000,抗体的半数抑制浓度(IC50)分别为281.6 ng/ml和98.4 ng/ml,与百草枯类似物没有明显交叉反应。结论合成的百草枯完全抗原具有较好的免疫原性,产生的抗百草枯多克隆抗体具有较好的灵敏度和特异性。

Object To modify paraquat（ PQ）hapten,to synthesize complete antigens,and to prepare anti-paraquat polyclonal antibody,so as to provide the foundation for rapid detection of PQ residue in food. Methods Paraquat was modified to be paraquat hapten（ PQ-hap）. Complete antigens,immunogen（PQ-BSA）and coating antigen（PQ-OVA）,were synthesized by coupling with carrier proteins（BSA and OVA）using mixture anhydrides method. The conjugates were identified by mass spectrography and SDS-PAGE. The polyclonal antibody was obtained by two rabbits＇ immunization,and the sensitivity and specificity of serum were evaluated by ELISA. Results The immunogen and coating antigen of paraquat were synthesized successfully. After the sixth immunization,the titre of antiserum from two rabbits reached 1∶ 40000,and 1∶ 160000respectively;and the half inhibition concentration（ IC50）was 281.6 ng /ml,and 98.4 ng/ml respectively.Both of the polyclonal antibodies had no conspicuous cross-reaction with the other analogues. Conclusion The paraquat complete antigen so synthesized has good immunogenicity and the polyclonal antibodies obtained are highly specific and sensitive.