猫杯状病毒抗原检测胶体金试纸条的研制  

Development of immune colloidal gold test strips for detecting feline calicivirus

作　　者：李华斌 田崇瑜 李欣昱 李春和 张然 杨峰清 刘媛 贺威 谭凌云 赵忠鹏 闫芳[1] LI Huabin;TIAN Chongyu;LI Xinyu;LI Chunhe;ZHANG Ran;YANG Fengqing;LIU Yuan;HE Wei;TAN Lingyun;ZHAO Zhongpeng;YAN Fang(College of Veterinary Medicine,Shanxi Agricultural University,Jinzhong,Shanxi 03080l,China;School of Basic Medicine Sciences,Anhui Medical University,Hefei 230032,China;Beijing Ximu Biotechonogy Co.,Ltd,Beijing 100176,China;Inner Mongolia Huaxi Biotechnology Co.,Ltd.,Huhhot 01011l,China)

机构地区：[1]山西农业大学动物医学学院,山西晋中030800 [2]安徽医科大学基础医学院,安徽合肥230032 [3]北京希牧生物技术有限公司,北京100176 [4]内蒙古华希生物科技有限公司,内蒙古呼和浩特010111

出　　处：《中国兽医学报》2023年第3期483-487,503,共6页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划子课题基金资助项目(2016YFD0501000);内蒙古自治区科技厅科技重大专项基金资助项目(201702097)。

摘　　要：以浓缩纯化的猫杯状病毒(feline calicivirus,FCV)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0融合,用间接ELISA试验检测细胞培养上清,获得5株阳性杂交瘤细胞克隆株,命名为1E2、4C3、5F11、6G9和7D2,分别制备腹水后进行纯化。以多克隆抗体为金标抗体,腹水ELISA和中和效价较高的单抗(4C3)作为检测线抗体,羊抗兔二抗为质检抗体,制备检测FCV病毒抗原的免疫胶体金快速诊断试纸条。在pH为7.4的条件下,金标抗体的最适质量浓度为0.1 g/L,检测线最佳包被质量浓度为1.0 g/L,质控线最终质量浓度为0.5 g/L。经检测该试纸条具有敏感性高,特异性强,稳定性好的特点,用制备的胶体金试纸条与RT-PCR方法同时对76份猫鼻咽拭子进行检测,比较两者的符合率,符合率在90%以上。结果表明,本试验研制的胶体金试纸条可以用于FCV的临床检测。The FCV virus strain was concentrated and purified,and then was used to immunize BALB/c mice.The spleen cells of immunized mice were fused with mouse myeloma cells SP2/0,and the culture supernatant of the fused cells was detected by indirect ELISA test.Five clones of hybridoma cells that stably secreted anti-FCV antibodies were obtained,they were named 1E2,4C3,5F11,6G9 and 7D2,respectively.In this experiment,the prepared polyclonal antibody was gold labeled antibody,ascites ELISA and monoclonal antibody(4C3)with high neutralization titer were used as detection line antibody,sheep anti-rabbit secondary antibody was used as quality inspection antibody,and the immunocolloidal gold rapid diagnosis test strip for detecting FCV virus antigen was prepared.Under the condition of pH7.4,the optimal concentration of gold standard antibody was 0.1 g/L,the optimal coating concentration of detection line was 1.0 g/L,and the final concentration of quality control line was 0.5 g/L.The test strip had the characteristics of high sensitivity,strong specificity and good stability.The prepared colloidal gold test strip and RT-PCR were used to detect 76 cat nasopharyngeal swabs at the same time.The coincidence rate between the two methods was more than 90%.This experiment shows that the colloidal gold test strip developed in this experiment can be used for clinical detection of feline calicivirus.

关 键 词：单克隆抗体 FCV 免疫胶体金 

分 类 号：S852.65[农业科学—基础兽医学] R392.33[农业科学—兽医学]