O型口蹄疫Cathay拓扑型病毒单抗制备及双抗体夹心ELISA方法的初步建立

doi:10.11843/j.issn.0366-6964.2024.09.025

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (9): 4012-4020.doi: 10.11843/j.issn.0366-6964.2024.09.025

O型口蹄疫Cathay拓扑型病毒单抗制备及双抗体夹心ELISA方法的初步建立

廖焕程(), 石正旺, 罗俊聪, 王婉莹, 冯露, 周静, 张帆, 石鑫泰, 田宏*()

中国农业科学院兰州兽医研究所兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室，兰州 730000

收稿日期:2023-10-13 出版日期:2024-09-23 发布日期:2024-09-27
通讯作者: 田宏 E-mail:l2440437014@163.com;xibeitian0931@163.com
作者简介:廖焕程(1999-)，男，河南信阳人，硕士，主要从事兽医学研究，E-mail: l2440437014@163.com
基金资助:
2022甘肃省创新联合体-家畜重要疫病诊断研制与产业化(22ZD6NA012);国家重点研发计划项目(2021YFD1800300);中央高校基本科研业务费专项资金资助;国家生猪技术创新中心(NCTIP-XD/C03);中国农业科学院创新计划(CAAS-CSLPDC-202302);2023农业技术产业体系建设(CARS-35);甘肃省科技重大专项计划(22ZD6NA001)

Preparation of Monoclonal Antibody against Cathay Topotype of FMDV Type O and Development of Double Antibody Sandwich ELISA for Cathay Topotype of FMDV Type O

Huancheng LIAO(), Zhengwang SHI, Juncong LUO, Wanying WANG, Lu FENG, Jing ZHOU, Fan ZHANG, Xintai SHI, Hong TIAN*()

State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China

Received:2023-10-13 Online:2024-09-23 Published:2024-09-27
Contact: Hong TIAN E-mail:l2440437014@163.com;xibeitian0931@163.com

摘要/Abstract

摘要：

本研究旨在纯化O型口蹄疫Cathay拓扑型病毒抗原，并制备单克隆抗体，为口蹄疫新型Cathay毒株的研究提供生物材料。PEG沉淀法纯化O型口蹄疫Cathay毒株抗原，免疫BALB/c小鼠，取脾细胞与SP2/0细胞融合制备杂交瘤细胞，用间接ELISA方法筛选阳性细胞，有限稀释法进行亚克隆，获得2株能够稳定分泌特异性针对O型口蹄疫Cathay株病毒单克隆抗体的杂交瘤细胞，分别命名为10E6与11C7。叠加试验表明，两株单抗的叠加率为49.45%，识别不同的抗原表位。间接ELISA和IFA试验显示，两株单抗与O型口蹄疫Cathay株病毒具有良好的反应性。单抗特异性检测结果表明，2株单抗均能特异性识别Cathay株口蹄疫病毒，不与其他口蹄疫病毒毒株交叉反应。抗体亚型鉴定结果显示，10E6单抗的轻链为Kappa链，11C7单抗的轻链为Lamda链，两株单抗重链类型均为IgG2a。结果表明，本研究成功制备了两株特异性结合O型口蹄疫Cathay拓扑型病毒的单克隆抗体，两株单抗均具有良好的反应性与特异性。应用两株单抗建立O型口蹄疫Cathay病毒的双抗体夹心ELISA检测方法，该ELISA检测方法特异性、重复性和敏感性良好，为O型口蹄疫Cathay毒株的快速诊断防控与研究建立了基础。

关键词: O型口蹄疫病毒, Cathay拓扑型, 杂交瘤细胞, 单克隆抗体, ELISA

Abstract:

This study aimed to purify the cathay topotype of foot-and-mouth disease virus (FMDV) type O and prepare monoclonal antibody to provide biological materials for the study of novel cathay strain of foot-and-mouth disease. The antigen of cathay strain of FMDV type O was purified by PEG precipitation method and immunized BALB/c mice. Hybridoma cells were prepared by fusion of spleen cells and SP2/0 cells. Positive cells were screened by indirect ELISA and subcloned by limited dilution method. Two hybridoma cells, named 10E6 and 11C7, which were able to secrete monoclonal antibodies specifically against O-type FMDV Cathay strain were obtained. Additivity assay showed that the additivity rate of the two mAbs was 49.45%, and different epitopes were identified.Indirect ELISA and IFA showed that the two strains had good reactivity with O-type FMDV Cathay strain. The results of monoclonal antibody detection showed that the two monoclonal antibodies could specifically recognize Cathay FMDV strains and did not cross-react with other FMDV strains. The results of antibody subtype identification showed that the light chain of 10E6 monoclonal antibody was Kappa chain, the light chain of 11C7 monoclonal antibody was Lamda chain, and the heavy chain type of both strains was IgG2a. The results showed that two monoclonal antibodies specifically binding to O-type FMDV Cathay topological strain were successfully prepared in this study. Both monoclonal antibodies had good reactivity and specificity. Two monoclonal antibodies were used to establish a double antibody sandwich ELISA method for detecting O-type FMDV Cathay strain. The ELISA method had good specificity, repeatability and sensitivity, which laid a foundation for the rapid diagnosis, control and research of O-type foot and mouth disease virus Cathay strain.

Key words: foot-and-mouth disease virus type O, Cathay topotype, hybridoma cells, monoclonal antibody, ELISA

中图分类号:

廖焕程, 石正旺, 罗俊聪, 王婉莹, 冯露, 周静, 张帆, 石鑫泰, 田宏. O型口蹄疫Cathay拓扑型病毒单抗制备及双抗体夹心ELISA方法的初步建立[J]. 畜牧兽医学报, 2024, 55(9): 4012-4020.

Huancheng LIAO, Zhengwang SHI, Juncong LUO, Wanying WANG, Lu FENG, Jing ZHOU, Fan ZHANG, Xintai SHI, Hong TIAN. Preparation of Monoclonal Antibody against Cathay Topotype of FMDV Type O and Development of Double Antibody Sandwich ELISA for Cathay Topotype of FMDV Type O[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4012-4020.

图/表 7

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OD_{450 nm}			AI/%
A_10E6	A_11C7	A_10E6+11C7	AI/%
1.73	1.91	2.72	49.45

项目 Item	O/Cathay	O/MYA98	AF/72	A/WH/09	对照组 Control
OD_{450 nm}	2.241	0.169	0.121	0.120	0.124
S/N	18.073	1.363	0.976	0.968	/

病毒悬液 Viral suspension	稀释倍数 Dilution ratio	批内重复性 Intra batch repeatability		批间重复性 Inter batch repeatability
病毒悬液 Viral suspension	稀释倍数 Dilution ratio	$\bar x \pm s$	变异系数/% CV	$\bar x \pm s$	变异系数/% CV
阳性样品	8×	16.19±0.27	1.68	16.02±0.35	2.18
Positive samples	16×	12.26±0.06	0.52	12.56±0.16	1.27
	32×	8.52 ± 0.42	4.94	8.12±0.52	6.40
阴性样品	8×	1.12±0.06	5.90	1.02±0.07	7.45
Negative samples	16×	0.95±0.06	6.71	0.93±0.07	7.93
	32×	0.68±0.05	7.53	0.70±0.06	8.73

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O型口蹄疫Cathay拓扑型病毒单抗制备及双抗体夹心ELISA方法的初步建立

Preparation of Monoclonal Antibody against Cathay Topotype of FMDV Type O and Development of Double Antibody Sandwich ELISA for Cathay Topotype of FMDV Type O

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