Establishment of an indirect ELISA method for detection of ECoV antibody in donkey and application
10.16303/j.cnki.1005-4545.2025.06.04
- VernacularTitle:间接ELISA检测驴源马冠状病毒抗体方法的建立与应用
- Author:
Yu YANG
1
;
Yu GUAN
;
Jiyuan LI
;
Chunyang YAO
;
Yanli BI
;
Leilei MO
;
Tongbin LI
;
Yueqiang XIAO
;
Heping ZHANG
Author Information
1. 河北工程大学生命科学与食品工程学院,河北邯郸 056021;山东省滨州畜牧兽医研究院,山东滨州 256600
- Publication Type:Journal Article
- Keywords:
donkey-derived equine coronavirus;
recombinant N protein;
indirect ELISA
- From:
Chinese Journal of Veterinary Science
2025;45(6):1126-1131
- CountryChina
- Language:Chinese
-
Abstract:
In order to establish a method for the detection of serum antibodies to donkey-derived e-quine coronavirus(ECoV),recombinant ECoV N protein was expressed in E.coli system,purified by nickel column affinity chromatography and identified by Western blot.After optimizing the re-action conditions,the indirect ELISA(iELISA)detection method was established using the puri-fied recombinant protein as coating antigen and used to detect 143 clinical serum samples.The re-sults showed that the recombinant N protein,which has good reaction activity with serum antibod-y,was successfully expressed.The optimum conditions of the established iELISA method were as follows:the amount of antigen coated was 0.2 μg/well and overnight at 4 ℃,10％skimmed milk powder solution was sealed at 37℃ for 1.5 h,the dilution concentration of serum was 1∶200,and the enzyme-labeled secondary antibody diluted at 1∶10 000.The sensitivity test results showed that the positive serum could be diluted to 1∶6 400.The specificity test results showed that all an-tibodies to several donkey pathogens were negative.The repetitive test results showed that the in-tra-and inter-batch coefficients of variation were 2.90％-6.12％and 2.29％-7.88％respectively.The positive rate of clinical donkey serum was 57.3％.The iELISA established in this study pro-vides a technical support for epidemiological investigation and antibody surveillance.
