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.
出处
《中国实验方剂学杂志》
CAS
北大核心
2013年第20期241-245,共5页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(30973961)
中南民族大学学术团队项目(XTZ09010)
关键词
剑叶龙血素A
人工抗原
多克隆抗体
酶联免疫吸附分析
cochinchinenin A
artificial antigen
polyclonal antibody
enzyme-linked immunosorbent assay