基于双特异纳米抗体FMDV血凝检测方法的建立

Establishment of haemagglutination detection method for quantification of foot and mouth disease virus based on bispecific nanobodies

本研究利用基因工程方法将抗口蹄疫病毒(FMDV)纳米抗体Nb205和抗鸡醛化红细胞(cRBC)纳米抗体NbRBC48的基因片段连接,构建双特异纳米抗体Nb205-48,其相对分子质量为3.5×10^(4),能同时与FMDV和cRBC反应。利用Nb205-48成功建立了可用于FMDV抗原检测的血凝方法,该方法检测灵敏度为1μg/ml,与其他病毒无交叉反应,且批内和批间重复性好。分别采用血凝法和夹心ELISA方法对3批次FMDV抗原进行了检测,二者检测结果基本吻合。本研究建立了基于双特异纳米抗体Nb205-48的血凝检测方法,该方法具有较好的灵敏度、特异性和重复性,且简便、快捷、成本低,其检测结果与传统的FMDV定量检测方法的检测结果相关性好,为检测口蹄疫疫苗生产过程中FMDV抗原的含量提供了新方法。

In this study,the gene fragments of the nanobody Nb205 against foot-and-mouth disease virus(FMDV)and the nanobody NbRBC48 against chicken aldehyde red blood cell(cRBC)were connected by genetic engineering method to construct the bispecific nanobody Nb205-48.Its relative molecular weight was 3.5×10^(4),and it could react with FMDV and cRBC simultaneously.A new hemagglutination method for the detection of FMDV antigen was established successfully by using Nb205-48.The detection sensitivity of this method was 1μg/ml,and it had no cross-reaction with other viruses.In addition,the intra-batch and inter-batch repeatability was good.Three batches of FMDV antigen were detected by hemagglutination method and sandwich ELISA method,and the results were similar.In this study,a novel hemagglutination method based on bispecific nanobody Nb205-48 was established.The method had good sensitivity,specificity and repeatability,and was simple,fast and low cost.The detection results were well correlated with the results of traditional FMDV quantitative detection methods.The method constructed in this study provides a new method for detecting the content of FMDV antigen in the production of foot-and-mouth disease vaccine.