RFFIT检测体系的改进和效果评估

发布时间：2019-01-01 18:41

【摘要】：目的: 1.对快速荧光灶抑制试验(rapid fluorescent focus inhibition test, RFFIT)检测体系进行本土化改进,以适应国内实验室条件和检测需求; 2.对改进后的RFFIT体系进行效果评估; 3.将本室已经应用成熟的RFFIT体系与狂犬病疫苗抗原检测方法进行融合,形成改良抗体结合试验(modified antibody binding test, M-ABT)。方法: 1.将BSR(BHK-21小克隆)、BHK-21(金黄地鼠肾细胞)、MNA(小鼠成神经纤维瘤细胞)三种细胞以及CVS-11、CTN-181两株狂犬病病毒分别引入RFFIT检测体系,以不同组合方式检测10份标本,初步鉴定检测结果的一致性;继而检测10~6份人血清样品,系统比较不同组合方式下RFFIT检测结果的差异。 2.对1011份人血清样品进行RFFIT检测,评估经过本土化改进的RFFIT检测体系的检测效能。 3.利用自行制备的Alexa 488标记抗狂犬病病毒核蛋白单克隆抗体对200份标本进行RFFIT检测,评估自主制备的抗狂犬病病毒核蛋白单克隆抗体进行RFFIT检测的效果。 4.将RFFIT与抗体结合试验(antibody binding test, ABT)方案融合,在疫苗样品稀释、剩余中和抗体检测、结果计算方面进行改进,形成改良抗体结合试验(M-ABT)。应用M-ABT对10种灭活狂犬病疫苗在不同库存时间的疫苗效力进行检测,观察疫苗效力随时间改变的情况。结果: 1.细胞培养:BSR、BHK-21、MNA细胞生长状况良好,建立了这3种细胞的细胞种子库; 2.病毒培养:狂犬病病毒CVS-11和CTN-181株对BSR、BHK-21、MNA细胞适应良好,各代次病毒滴度均高于10~6 FFU/ml,符合RFFIT检测体系对毒种的要求; 3.改变细胞系和毒种后RFFIT检测体系的检测效能评估:10份小批量样本检测:分别引进BHK-21和MNA细胞系以及CTN-181病毒株后,RFFIT检测结果与采用BSR和CVS-11组合进行检测所得结果间差别无统计学意义,P值均大于0.05;106份大批量样本的检测结果也表明引入新的细胞系和毒株后RFFIT检测体系与国际标准体系检测结果间差别无统计学意义,并且有很好的相关关系,相关系数(r)大于0.75。 4.改进RFFIT检测体系应用效果评估: ⑴不同细胞系对RFFIT检测结果的影响:以CVS-11病毒株作为攻击病毒,分别应用BSR、BHK-21、MNA细胞系,对1011份人血清样品进行RFFIT检测,不同检测体系的阳性检出率差别无统计学意义,P值均大于0.05;不同检测体系下阳性标本检测结果几何均值(geometric mean titer, GMT)差别无统计学意义,P值均大于0.05;不同检测体系下检测结果相关性较好,相关系数r大于0.75; ⑵不同操作环境对RFFIT检测结果的影响:在不同地理位置、操作环境迥异的实验室,由相同的操作人员应用CVS-11毒株和BHK-21细胞系对上述阳性人血清标本再次进行RFFIT检测,检测结果与原实验室环境下进行检测所得结果间差别无统计学意义,P值均大于0.05。 5.采用自主制备Alexa 488标记抗狂犬病病毒核蛋白单克隆抗体后RFFIT检测体系检测效能评估:对200份血清样品的检测显示采用自主制备Alexa 488标记抗狂犬病病毒核蛋白单克隆抗体与进口单抗Millipore DFA 5100检测结果差别无统计学意义,相关系数r =0.980。 6. M-ABT法的建立和初步应用效果分析:10种待测狂犬病疫苗经M-ABT法检测,结果显示疫苗中抗原含量与NIH法测定结果差别没有统计学意义,并且具有很好的相关关系,相关系数r=0.927,提示M-ABT法可用于监测狂犬病疫苗抗原的效价变化情况。结论: 1.实现了应用不同细胞系和狂犬病病毒株进行RFFIT检测:将致病性更弱的CTN-181毒株应用于狂犬病病毒中和抗体的实验室检测,增加了检测安全性;使用常见的BHK-21、MNA细胞系使更多实验室建立和应用该检测技术成为可能; 2.不同操作环境对RFFIT检测结果的影响不明显,因此,同一批操作人员使用相同的检测体系在不同实验室进行RFFIT检测的结果均衡可比并可信; 3.自主制备的Alexa488标记抗狂犬病核蛋白单克隆抗体可以用于国内RFFIT检测; 4. M-ABT法已经建立,用于灭活狂犬病疫苗效力检测时与NIH法检测结果高度一致,是一种合适的灭活狂犬病疫苗效力监测技术手段。
[Abstract]:Purpose: 1. The localization of the rapid fluorescence focus detection test (RFIIT) detection system is improved to meet the domestic laboratory conditions and detection requirements.? 2. Effect on the improved RFIIT system Fruit evaluation; 3. The room has been fused with a mature RFIIT system and a rabies vaccine antigen detection method to form modified antibody binding test, M -AB Methods: 1. The BSR (BHK-21 small clone), BHK-21 (hamster kidney cell), MNA (mouse fibroblast cell) and CVS-11 and CTN-181 rabies virus were introduced into the RFFIT detection system. the consistency of the detection results is determined; in turn, 10 to 6 human serum samples are detected, and the system is compared with the RFF in different combinations; The difference in the IT test results. 2. The RFFIT test was performed on 1011 human serum samples to assess the localization of the modified RFF the detection efficiency of the IT detection system is 3. 200 samples of the anti-rabies virus nucleoprotein monoclonal antibody prepared by using the self-prepared Alexa 488-labeled anti-rabies virus nucleoprotein monoclonal antibody are used for detecting and evaluating the self-prepared anti-rabies virus nucleoprotein monoclonal antibody. 4. The RFIIT was fused with the antibody binding assay (ABT), and the results were improved and formed in the sample dilution, the remaining neutralizing antibody test and the calculation of the results. Improved antibody binding assay (M-ABT). Detection of the efficacy of 10 inactivated rabies vaccines in different stock times with M-ABT, observe Results: 1. Cell culture: BSR, BHK-21, MNA cell growth In good condition, the cell seed bank of the three cells was established; 2. Virus culture: The rabies virus CVS-11 and the CTN-181 strain were well adapted to the BSR, BHK-21, and MNA cells. and the drop degree of each generation virus is higher than 10-6FFU./ ml, in line with the requirements of the RFFIT detection system for the virus species; 3. Evaluation of the detection efficacy of the RFIIT detection system after the change of the cell line and the virus species: 10 small batch sample testing: introduction of BHK, respectively After the 21 and MNA cell lines and the CTN-181 strain, the results of the RFFIT test were not statistically significant with the results obtained by the combination of the BSR and the CVS-11, and the P-value was greater than 0.05; the results of the detection of 106 large-scale samples also showed that the introduction of new cell lines and the post-strain RFFIT test There is no statistical significance between the test system and the international standard system test results, and and the correlation coefficient (r) is greater than 0.. 75. 4. Improvement of the application effect evaluation of the RFIIT detection system: The effect of different cell lines on the results of RFFIT detection: The CVS-11 virus strain is used as the attack virus, and the BSR and the BHK-21 are respectively applied. In the MNA cell line, 1011 human serum samples were tested by RFFIT. The positive rate of positive detection in different detection systems was not statistically significant, and the P value was greater than 0.05. The geometric mean (GMT) difference of positive samples under different detection systems was not statistically significant. and the P value is greater than 0.05; no The correlation of the test results with the detection system is good, the correlation coefficient r is greater than 0.75, and the influence of different operating environments on the RFFIT detection results is that the CVS-11 strains and the B are applied by the same operators in different geographic locations and different operating environments In the HK-21 cell line, the above-mentioned positive human serum samples were subjected to RFFIT detection, and the results of the test and the original laboratory ring The difference between the results obtained in the context of the detection was not statistically significant, and the P value was greater than 0.05. 5. The self-prepared Alexa 488 standard was used. Evaluation of the efficacy of the RFIIT detection system after the anti-rabies virus nucleoprotein monoclonal antibody: The detection of 200 serum samples shows the use of the self-prepared Alexa 488-labeled anti-rabies virus nucleoprotein monoclonal antibody and the imported monoclonal antibody Millipore The difference of DFA 5100 was not statistically significant, and the correlation coefficient r = 0.980. 6.M-ABT method was established and the preliminary application results were analyzed: 10 rabies vaccines to be tested were tested by M-ABT method, and the results showed that the antigen content in the vaccine and the results of NIH method The difference was not statistically significant and had a good correlation, and the correlation coefficient r = 0. 9 27. It is suggested that the M-ABT method can be used to monitor the change of the titer of the rabies vaccine antigen. the detection safety is increased by the laboratory detection of the rabies virus and the antibody, Using the common BHK-21, the MNA cell line makes it possible to establish and apply the detection technique to more laboratories; 2. The effect of different operating environments on the RFFIT detection results is not clear, and therefore, the same The same test system was used by the batch operators to balance the results of the RFFIT detection in different laboratories and be credible; The self-prepared Alexa488-labeled anti-rabies nucleoprotein monoclonal antibody can be used for domestic RFFIT detection; 4.M-ABT method has been established for killing live rabies
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【相似文献】