Preparation of monoclonal antibody against σA protein of avian reovirus and es-tablishment of sandwich ELISA method for detection
10.16303/j.cnki.1005-4545.2024.07.05
- VernacularTitle:禽呼肠孤病毒σA蛋白单克隆抗体的制备及夹心ELISA检测方法的建立
- Author:
Bingyi YANG
1
,
2
;
Zhixun XIE
;
Zhiqin XIE
;
Hongyu REN
;
You WEI
;
Liji XIE
;
Jiaoling HUANG
;
Sheng WANG
Author Information
1. 广西大学动物科学技术学院,广西南宁 530004
2. 广西壮族自治区兽医研究所广西兽医生物技术重点实验室,广西南宁 530001
- Keywords:
avian reovirus;
σA protein;
monoclonal antibody;
sandwich ELISA
- From:
Chinese Journal of Veterinary Science
2024;44(7):1373-1379
- CountryChina
- Language:Chinese
-
Abstract:
In order to prepare monoclonal antibody to σ A protein of avian reovirus(ARV)and es-tablish a sandwich ELISA method for the detection of ARV pathogens.In this study,the σ A pro-tein of ARV was expressed as antigen by prokaryotic expression and used to immunize BALB/c mice.Then,stable hybridoma cell lines were screened,and monoclonal antibodies were prepared.A sandwich ELISA detection method based on monoclonal antibody of σA protein was established,and the sensitivity,specificity,repeatability,and accuracy were tested.The results showed that the recombinant plasmid pET-32a-σA was successfully constructed and well expressed in Escherichia coli.After immunizing mice,two hybridoma cell lines 6B3 and 8E11,which could secrete mono-clonal antibodies stably,were successfully prepared.Both monoclonal antibodies could react with natural ARV.One of the monoclonal antibodies secreted by 6B3 was selected as the capture anti-body and the ARV-positive chicken polyclonal antibody was used as the detection antibody.A sand-wich ELISA method was established to detect ARV by optimizing the reaction conditions.The specific test showed that the method only detected ARV pathogens and no other common chicken viral pathogens were detected.The detection limit was 7.72 X 102 EID50/mL of ARV antigen.The coefficient of variation of the intra-and inter-assay tests were less than 5.0％and the reproducibili-ty was good.Thirty samples were tested simultaneously by σA-sandwich ELISA and PCR,and the results were consistent with each other.In conclusion,a sandwich ELISA method based on the monoclonal antibody of σA protein was successfully established for the identification and detection of ARV,which provided a technical means for the accurate and rapid detection of ARV.
