体外检测试剂盒阻断剂的制备方法及特性分析  

Preparation and characteristics analysis of blockers for in vitro diagnostic test kits

作　　者：何汝琴 雷鹏 邓龙芝 朱文武 朱雄伟 熊梦瑶 He Ruqin;Lei Peng;Deng Longzhi;Zhu Wenwu;Zhu Xiongwei;Xiong Mengyao(School of Environmental Ecology and Bioengineering,Wuhan Institute of Technology,Hubei,Wuhan 430205,China;Wuhan Yesda Biotechnology Co.,Ltd,Hubei,Wuhan 430200,China)

机构地区：[1]武汉工程大学环境生态与生物工程学院,湖北武汉430205 [2]武汉雁达生物技术有限公司,湖北武汉430200

出　　处：《发育医学电子杂志》2024年第1期1-6,19,共7页Journal of Developmental Medicine (Electronic Version)

基　　金：国家自然科学基金(42376122)。

摘　　要：目的探讨体外检测试剂盒阻断剂的制备方法及特性。方法采用6周龄BALB/c小鼠进行抗原免疫,构建杂交瘤细胞并克隆。采用动物体内诱导单克隆抗体的方法大量制备单克隆抗体,分别采用G柱亲和层析、蓝胶+Q柱离子交换层析两种方法进行纯化,核酸蛋白检测仪记录波长280 nm下的光密度(optical density,OD),紫外分光光度计测量成品浓度。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecylsulfate-polyacrylamide gel electrophoresis,SDS-PAGE)检测抗体纯度。采用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)双抗体夹心法检测该阻断剂(CN302)对假阳性样本的阻断能力。结果G柱亲和层析法提取阻断剂的收率(3个批次分别为6.49、6.12、6.27 g/L)高于蓝胶+Q柱离子交换层析法(3个批次分别为1.78、1.68、1.61 g/L)。两种纯化方法的纯度均高达95%以上。G柱亲和层析法、蓝胶+Q柱离子交换层析法所得阻断剂的活性分别为106.54%、92.45%;与空白组相比,两种纯化方式所得阻断剂CN302对假阳性样本的检出率均降低,其OD值均小于0.2,与基准阻断剂大致相同。G柱亲和层析法所得阻断剂CN302未冻融的活性为99.86%,冻融5次后活性为99.83%。无阻断剂存在时,对假阳性样本检测的OD值约为2.5,最高可达到4.0;加入阻断剂后,OD值明显降低;阻断剂CN302对假阳性样本检测的OD值低于其他阻断剂。结论本研究所得阻断剂能显著消除样本内源性干扰,且活性几乎全部保留。G柱亲和层析法提取抗体的收率和阻断能力均高于蓝胶+Q柱离子交换层析法。Objective To explore the preparation methods and characteristics of blockers for in vitro diagnostic test kits.Method Six-week-old BALB/c mice were used for antigen immunization.Hybridoma cells were constructed and cloned.The method of inducing monoclonal antibodies in animal bodies was used to prepare a large quantity of monoclonal antibody,which was purified by two methods(G-column affinity chromatography,and blue gel+Q-column ion-exchange chromatography).The optical density(OD)was recorded at a wavelength of 280 nm using a nucleic acid protein detector.Finished product concentration was detected using a ultraviolet spectrophotometer.The purity of the antibody was detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Enzyme-linked immunosorbent assay(ELISA)double antibody sandwich method was used to detect the blocking ability of this blocker(CN302)on false positive samples.Result The yields of the blocker extracted by G-column affinity chromatography(6.49,6.12,and 6.27 g/L for three batches,respectively)were higher than those by blue gum+Q-column ion-exchange chromatography(1.78,1.68,and 1.61 g/L for three batches,respectively).The purity of both purification methods was up to more than 95%.The activities of the blockers obtained by G-column affinity chromatography and blue gum+Q-column ion-exchange chromatography were 106.54%and 92.45%,respectively.Compared with the blank group,the detection of false-positive samples was reduced by blockers extracted using both purification methods,and the OD values of the blocker CN302 were less than 0.2,which were about the same as that of the reference blocker.The activity of the blocker CN302 obtained by G-column affinity chromatography was 99.86%without freezing and thawing,and 99.83%after 5 times of freezing and thawing.Without blocker,the OD values of false-positive samples were about 2.5 and up to 4.0,that decreased significantly after the addition of the blocker.The OD values of the blocker CN302 were lower than those of the other blockers fo

关 键 词：阻断剂 体外检测试剂盒 小鼠 单克隆抗体 G柱亲和层析 蓝胶+Q柱离子交换层析 

分 类 号：TH789[机械工程—仪器科学与技术]