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

摘要:

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12 000。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,IC₅₀ 0.66 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。

Abstract:

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.