剑叶龙血素A间接竞争ELISA检测方法的建立

Development of Indirect Competitive ELISA for the Detection of Cochinchinenin A

目的：建立间接竞争酶联免疫吸附分析法（ELISA）用于测定剑叶龙血素A（CA）含量.方法：合成半抗原剑叶龙血素A-4＇-羧甲基醚（CA-4＇-CME）,再用碳二亚胺法将其与牛血清白蛋白（BSA）和鸡卵清蛋白（OVA）分别偶联制备免疫原（CA-BSA）和包被原（CA-OVA）,免疫小鼠制备多克隆抗体,建立间接竞争ELISA法并评估其分析性能.结果：通过紫外扫描分析CA-BSA和CA-OVA的偶联比分别为18∶1和24∶1.抗血清效价最高达到12000.用四参数logistic方程拟合CA标准曲线,在分析范围0 ～0.80 mg·L-1内,相关系数（R2）≥0.99,IC500.66 mg·L-1,最低检测限0.045 mg·L-1,批内CV≤12.5％,批间CV≤14.7％,回收率92.7％ ～ 118.7％,对5种CA类似物的交叉反应率＜5.38％.此方法检测龙血竭和龙血竭总黄酮中CA含量分别为1.27％,3.08％.结论：剑叶龙血素A间接竞争ELISA法精密度、准确度和特异性均较好,可方便地用于药物检测和分析.

Objective：To establish an indirect competitive enzyme-linked immunosorbent assay （ELISA） for the determination of Cochinchinenin A （CA）.Method：Cochinchinenin A-4＇-Carboxymethyl ether （CA-4＇CME） was synthesized as hapten.The hapten was attached to Bovine serum albumin （BSA） and ovalbumin （OVA） as artificial antigen by carbodimide method.Anti-CA polyclonal antibody was obtained from mice which were immunized with CA-BSA.A method of the indirect competitive ELISA for CA was established,and the analytical performance of this method was evaluated.Result：The conjugation molar ratio of CA to BSA and OVA were 18∶1 and 24∶ 1,respectively,calculated by UV spectrophotometry.The highest titer of Anti-CA sera was 12 000.Standard curve of CA was fitted by four-parameter logistic equation.In the analysis range of 0-0.80 mg ·L-1,the correlation coefficient was larger than 0.99.The IC50 and LOD of the method were 0.66 mg ·L-1 and 0.045 mg ·L-1,respectively.The intra and inter assay precisions were less than 12.5％ and 14.7％ respectively.And recoveries ranged from 92.7％-118.7％ in typical matrixes.The cross-reactivities of five structural analogues of CA were less than 5.38％.Detected by this method,the mass proportion of CA in Chinese Dragon＇s blood and total flavonoids of Chinese Dragon＇s blood were 1.27％,3.08％ respectively.Conclusion：Indirect competitive ELISA for the determination of CA,with its high sensitivity,specificity and accuracy,can be conveniently used in drug testing and analysis.