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目的合成绿原酸(chlorogenic acid,CHA)人工抗原,制备CHA多克隆抗体,为制备过敏成分分析的免疫芯片奠定基础。方法采用碳二亚胺法分别合成CHA免疫抗原(CHA-牛血清白蛋白)和包被抗原(CHA-卵清蛋白);UV法和TLC法鉴定人工抗原偶联是否成功;间接ELISA方法检测免疫小鼠血清中抗体效价;间接竞争ELISA方法检测抗体特异性。结果经UV法和TLC法检测,CHA与BSA偶联成功。血清抗体效价为1∶128 000,CHA-PAbs的质量浓度与抑制率的线性方程为Y=-0.10 lnX+1.158,线性检测范围为15.6~250 ng/mL,R2=0.995,并且和甘草酸、黄芩苷等与其常配伍应用的中药化合物均无交叉反应。结论成功制备了灵敏度高、特应性好的CHA多克隆抗体,为CHA的微量检测和过敏性研究提供了实验依据。
Objective To synthesize CHA artificial antigen and prepare CHA polyclonal antibody, which will lay the foundation for the preparation of immune chip for allergy analysis. Methods The CHA-immunized antigen (CHA-bovine serum albumin) and the coated antigen (CHA-ovalbumin) were synthesized by carbodiimide method. The conjugation of artificial antigen was identified by UV and TLC. Antibody titers in mouse serum; indirect competitive ELISA method to detect antibody specificity. Results The results of UV and TLC showed that CHA and BSA were successfully coupled. The titer of serum antibody was 1:128 000, the linear equation of mass concentration and inhibition rate of CHA-PAbs was Y = -0.10 lnX + 1.158, the linear range was 15.6-250 ng / mL, R2 = 0.995, , Baicalin and other often used in traditional Chinese medicine compound cross-reaction. Conclusion The polyclonal antibody against CHA with high sensitivity and good specificity has been successfully prepared and provided an experimental basis for the trace detection and allergy research of CHA.