半夏凝集素蛋白单克隆抗体的制备及双抗夹心ELISA的建立  

Preparation of monoclonal antibodies against Pinellia ternata lectin protein and establishment of double-antibody sandwich ELISA

作　　者：谢雨薇 郁红礼[1,2,3] 吴皓[1,2,3] 陶兴宝 王贺鹏 程砚秋 王彩霞 曾平 刘冰冰 张萍[4] 崔小兵[1] XIE Yu-wei;YU Hong-li;WU Hao;TAO Xing-bao;WANG He-peng;CHENG Yan-qiu;WANG Cai-xia;ZENG Ping;LIU Bing-bing;ZHANG Ping;CUI Xiao-bing(School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Jiangsu Key Laboratory of Chinese Medicine Procssing,Nanjing 210023,China;Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing,Nanjing 210023,China;National Institutes for Food and Drug Control,Beijing 100050,China)

机构地区：[1]南京中医药大学药学院,江苏南京210023 [2]江苏省中药炮制重点实验室,江苏南京210023 [3]国家教育部中药炮制规范化及标准化工程研究中心,江苏南京210023 [4]中国食品药品检定研究院,北京100050

出　　处：《中国中药杂志》2022年第22期6076-6081,共6页China Journal of Chinese Materia Medica

基　　金：国家重点研发计划项目(2018YFC1707000);国家自然科学基金项目(81173549)。

摘　　要：为测定半夏及其炮制品中内源性毒性物质半夏凝集素蛋白(Pinellia ternata lectin, PTL)的含量,该文采用杂交瘤细胞技术制备PTL特异性单克隆抗体,并建立了PTL抗原定量双抗夹心ELISA检测方法,检测条件为:捕获抗体工作浓度为2.5μg·mL-1,检测抗体稀释倍数为1∶450,包被条件为4℃过夜,封闭时间、抗原及检测抗体孵育时间均为90 min,辣根过氧化物酶标记链霉亲和素(streptavidin-horseradish peroxidase, SA-HRP)孵育时间为15 min。该方法对PTL抗原的定量限为0.375 ng·mL-1;线性范围为75.000~4 800.000 pg·mL-1,R~2=0.997 1;加样回收率在90.0%~110.0%;试验内和试验间精密性变异系数分别在2.0%~3.0%、2.0%~8.5%。采用该方法测定3批半夏及其炮制品中PTL含量,测得半夏中PTL质量分数均值为35.42 mg·g^(-1),其炮制品清半夏、法半夏、姜半夏中PTL质量分数均值分别为1.15 mg·g^(-1)、16.53μg·g^(-1)和122.63 ng·g^(-1),表明炮制后PTL含量显著下降。该文建立的PTL抗原定量双抗夹心ELISA检测方法线性较好,反应灵敏,准确度高,为半夏炮制过程中PTL含量检测提供一种简便有效的监测方法。To determine the content of endogenous toxic substance Pinellia ternata lectin(PTL) protein in Pinelliae Rhizoma and the related processed products, this study prepared specific monoclonal antibodies against PTL by hybridoma cell technology, and established a quantitative double-antibody sandwich enzyme linked immunosorbent assay(ELISA) for PTL antigen. The detection conditions were 2.5 μg·mL-1working concentration of the captured antibody and 1∶450 of the dilution multiple of detected antibody. The coating condition was staying overnight at 4 ℃. The blocking time and incubation times of antigen and detected antibody were all 90 minutes. The incubation time of horseradish peroxidase conjugated streptavidin-horseradish peroxidase(SA-HRP) was 15 minutes. The quantitative limit of the method for PTL antigen was 0.375 ng·mL-1. The linear range was 75.000-4 800.000 pg·mL-1, and R~2=0.997 1. The recovery rate was 90.0%-110.0%, and the variation coefficients of intra-test and inter-test precision were 2.0%-3.0% and 2.0%-8.5%.The content of PTL in three batches of Pinelliae Rhizoma and the related processed products was determined by the method, and the average content of PTL in Pinelliae Rhizoma was 35.42 mg·g^(-1). The average content of PTL in Pinelliae Rhizoma Praeparatum Cum Alumine, Pinelliae Rhizoma Praeparatum, and Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumine were 1.15 mg·g^(-1), 16.53 μg·g^(-1), and 122.63 ng·g^(-1), respectively, indicating that the content of PTL decreased significantly after processing. The quantitative double-antibody sandwich ELISA for PTL antigen established in this paper had good linearity, sensitive response, and high accuracy, which provided a simple and effective monitoring method for the detection of PTL content in the processing of Pinelliae Rhizoma.

关 键 词：半夏 凝集素蛋白 单克隆抗体 双抗夹心ELISA 

分 类 号：R284.1[医药卫生—中药学]