荧光标记抗狂犬病病毒核蛋白单克隆抗体的制备及初步应用
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摘要
抗狂犬病病毒(Rabies virus,RABV)核蛋白荧光抗体具有价格昂贵、保质期短和购买周期长等缺点,非常不利于各级疾病预防控制系统对该试剂的紧急使用。作为国家狂犬病实验室,亟需研发具有自主知识产权的异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)标记抗RABV核蛋白单克隆抗体(Monoclonal antibody,MAb),并且商品化,使其广泛应用于流行病学调查和实验室的病原学和血清学诊断,实现国家狂犬病监测和检测相关试剂的标准化,从而为我国狂犬病防控提供技术支持。制备FITC标记的抗RABV核蛋白MAb,并将其初步应用于直接免疫荧光实验(Direct fluorescent assay,DFA)和快速荧光灶抑制试验(Rapid fluorescent focus inhibition test,RFFIT)。将本实验室制备的抗RABV核蛋白MAb 3B12进行FITC标记,通过DFA确定荧光标记抗体(FITC--3B12)工作浓度,以市售FITC标记MAb作为阳性对照,对狂犬病相关临床样本进行DFA和RFFIT检测。DFA实验结果显示,FITC--3B12的工作浓度为1∶80,与市售荧光抗体的检测结果符合率为100%。RFFIT结果显示,FITC-3B12与市售荧光抗体的检测结果无统计学差异,且具有良好的重复性(CV<15%)。以上结果表明,本实验室所制备的抗RABV核蛋白荧光抗体可应用于狂犬病相关的DFA及RFFIT检测。
The fluorescein isothiocyanate(FITC) labeled anti-rabies virus(RABV) nucleoprotein monoclonal antibody(MAb) has the characteristics of high price, short quality guarantee period but long purchase period,which is very unfavorable for the emergency use of the reagent. As the national rabies laboratory, it is urgent to develop a F-ITC labeled anti-RABV nucleoprotein MAb with proprietary intellectual property rights, which can be widely used in epidemiological investigations and pathogenic and serological diagnosis of laboratory. This is beneficial for rabies surveillance and standardization of related reagents, and provides technical support for prevention and control of rabies in China. To prepare MAbs against RABV nucleoprotein labeled with FITC and preliminarily apply them to direct fluorescent assay(DFA) and rapid fluorescent focus inhibition test(REFIT),the MAb 3B12 against RABV nucleoprotein prepared in our laboratory was labeled with FITC. The working concentration of the labeled FITC-3B12 was determined by DFA. The commercial FITC-labeled MAb was used as positive control. The clinical samples related to rabies were detected by DFA and RFFIT, respectively.DFA results showed that the working concentration of FITC-3B12 was 1: 80, and the coincidence rate between the detection results of FITC-3B12 and those of commercial fluorescent antibodies was 100%. The results of RFFIT showed that there was no significant difference between the fluorescent antibodies prepared in this study and the commercial FITC-labeled MAb,and they had good repeatability(CV< 15%). The results indicated that the fluorescent antibody against RABV nucleoprotein(FITC-3B12) prepared in our laboratory can be applied in the rabies-related detection of DFA and RFFIT.
The fluorescein isothiocyanate(FITC) labeled anti-rabies virus(RABV) nucleoprotein monoclonal antibody(MAb) has the characteristics of high price, short quality guarantee period but long purchase period,which is very unfavorable for the emergency use of the reagent. As the national rabies laboratory, it is urgent to develop a F-ITC labeled anti-RABV nucleoprotein MAb with proprietary intellectual property rights, which can be widely used in epidemiological investigations and pathogenic and serological diagnosis of laboratory. This is beneficial for rabies surveillance and standardization of related reagents, and provides technical support for prevention and control of rabies in China. To prepare MAbs against RABV nucleoprotein labeled with FITC and preliminarily apply them to direct fluorescent assay(DFA) and rapid fluorescent focus inhibition test(REFIT),the MAb 3B12 against RABV nucleoprotein prepared in our laboratory was labeled with FITC. The working concentration of the labeled FITC-3B12 was determined by DFA. The commercial FITC-labeled MAb was used as positive control. The clinical samples related to rabies were detected by DFA and RFFIT, respectively.DFA results showed that the working concentration of FITC-3B12 was 1: 80, and the coincidence rate between the detection results of FITC-3B12 and those of commercial fluorescent antibodies was 100%. The results of RFFIT showed that there was no significant difference between the fluorescent antibodies prepared in this study and the commercial FITC-labeled MAb,and they had good repeatability(CV< 15%). The results indicated that the fluorescent antibody against RABV nucleoprotein(FITC-3B12) prepared in our laboratory can be applied in the rabies-related detection of DFA and RFFIT.
引文
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[4]Pengcheng Yu,Xinjun Lyu,Xinxin Shen,Qing Tang,Guodong Liang.Establishment and preliminary applica-tion of a rapid fluorescent focus inhibition test(RFFIT)for rabies virus[J].Virol Sin,2013,28(4):223-227.
[5]周航,李昱,陈瑞丰,陶晓燕,于鹏程,曹守春,李丽,陈志海,朱武洋,殷文武,李玉华,王传林,余宏杰.狂犬病预防控制技术指南(2016版)[J].中华流行病学杂志,2016,37(2):139-163.
[6]唐佳佳,李小兵,刘国文,孔涛,刘磊,杨威,王哲.单克隆抗体纯化的研究进展[J].中国畜牧兽医,2011,38(2):76-80.
[7]肖增鸿,黄昭亮,林月霞,董斌,田素娟.腹水型单克隆抗体纯化方法的研究[J].中国医药生物技术,2013,6:425-428.
[8]徐志伟,邓鸿业,韩淑红,邓玉兰,黄大无,丁桂凤.FITC标记的抗Brdu单克隆抗体的研制[J].细胞与分子免疫学杂志,2000,16(2):164-165.
[9]潘玉民,董玉平,石全瑞,辛英.小鹅瘟免疫荧光诊断方法的研究[J].中国兽医科技,1990,20(10):8-12.
[10]Goldman M.Fluorescent antibody methods[M].New York:Academic Press,1968:149.
[11]严加新,祝玉桃,李成平,徐葛琳,朱家鸿.抗狂犬病毒和蛋白荧光标记抗体的制备和初步鉴定[J].中国人兽共患病杂志,2000,16(5):59-62.
[2]WHO Expert Consultation on Rabies:third report[DB].https://apps.who.int/iris/bitstream/handle/10665/272364/9789241210218-eng.pdf?ua=1
[3]Wenjuan Wang,Xiong Li,Lihua Wang,Hu Shan,Lei Cao,Pengcheng Yu,Qing Tang,Guodong Liang.Prep-aration and identification of anti-rabies virus monoclonal antibodies[J].Virol Sin,2012,27(3):172-178.
[4]Pengcheng Yu,Xinjun Lyu,Xinxin Shen,Qing Tang,Guodong Liang.Establishment and preliminary applica-tion of a rapid fluorescent focus inhibition test(RFFIT)for rabies virus[J].Virol Sin,2013,28(4):223-227.
[5]周航,李昱,陈瑞丰,陶晓燕,于鹏程,曹守春,李丽,陈志海,朱武洋,殷文武,李玉华,王传林,余宏杰.狂犬病预防控制技术指南(2016版)[J].中华流行病学杂志,2016,37(2):139-163.
[6]唐佳佳,李小兵,刘国文,孔涛,刘磊,杨威,王哲.单克隆抗体纯化的研究进展[J].中国畜牧兽医,2011,38(2):76-80.
[7]肖增鸿,黄昭亮,林月霞,董斌,田素娟.腹水型单克隆抗体纯化方法的研究[J].中国医药生物技术,2013,6:425-428.
[8]徐志伟,邓鸿业,韩淑红,邓玉兰,黄大无,丁桂凤.FITC标记的抗Brdu单克隆抗体的研制[J].细胞与分子免疫学杂志,2000,16(2):164-165.
[9]潘玉民,董玉平,石全瑞,辛英.小鹅瘟免疫荧光诊断方法的研究[J].中国兽医科技,1990,20(10):8-12.
[10]Goldman M.Fluorescent antibody methods[M].New York:Academic Press,1968:149.
[11]严加新,祝玉桃,李成平,徐葛琳,朱家鸿.抗狂犬病毒和蛋白荧光标记抗体的制备和初步鉴定[J].中国人兽共患病杂志,2000,16(5):59-62.
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