Preparation of monoclonal antibodies against DHAV-3 3D protein and establish-ment of DAS-ELISA detection method
10.16303/j.cnki.1005-4545.2024.12.08
- VernacularTitle:DHAV-3 3D蛋白单克隆抗体制备及其DAS-ELISA检测方法的建立
- Author:
Xudong FENG
1
;
Linnan WU
;
Tianze CHEN
;
Mengru ZHAO
;
Yanyan LIU
;
Xuehui ZHOU
;
Xiaowei YANG
;
Lei YU
;
Liwu ZHANG
;
Guangwei ZHAO
Author Information
1. 西南大学动物医学院,重庆荣昌 402460
- Publication Type:Journal Article
- Keywords:
DHAV-3;
monoclonal antibodies;
DAS-ELISA;
3D protein;
detection method
- From:
Chinese Journal of Veterinary Science
2024;44(12):2556-2563,2578
- CountryChina
- Language:Chinese
-
Abstract:
In order to achieve rapid detection of duck hepatitis A virus type 3(DHAV-3),this ex-periment initially performed prokaryotic expression of the non-structural protein 3D of DHAV-3,followed by immunization of BALB/c mice with the purified protein.After immunization,mouse spleen cells were fused with myeloma cells(SP2/0)to prepare monoclonal antibodies.Subsequent-ly,a double-antibody sandwich ELISA(DAS-ELISA)detection method was established using the monoclonal antibodies,and its sensitivity,specificity,and repeatability were evaluated.Finally,the established method was applied to the detection of clinical samples and validated for compliance with the RT-PCR method.The results showed that the DHAV-3 3D protein was efficiently ex-pressed in BL21(DE3),and its specificity was confirmed by Western blot after purification.After cell fusion and three rounds of subcloning,six hybridoma cells were successfully screened and named 1A3,1B6,1C7,1D9,2A1,and 3A9.The subtype identification of the antibodies showed that 1A3 belonged to IgG2b,1B6 belonged to IgG2a,3A9 belonged to IgG3,and 1C7,1D9,and 2A1 be-longed to IgG1.After screening,the high-affinity monoclonal antibodies 1B6 and 1 A3 were selected as the capture antibody and detection antibody,respectively,and use to establish the DAS-ELISA detection method.After optimizing the reaction conditions,the optimal coating concentration of the capture antibody 1B6 was determined to be 1×10-3 g/L,and the optimal dilution of the detection antibody 1A3 was 1∶1 000.The cut-off value was established as 0.256.The sensitivity test showed that the method had a minimum detection limit of 4.0 ×10-4 g/L for the 3D protein.The repeat-ability test showed that the within-batch and between-batch coefficients of variation were both less than 9％,indicating good repeatability.The specificity test showed that the method did not show specific reactions with duck adenovirus(DAdV),muscovy duck parvovirus(MDPV),duck circo-virus(DuCV),duck plague virus(DPV),duck reovirus(DRV),or Riemerella anatipestifer(RA),but cross-reacted with Duck hepatitis a virus type 1(DHAV-1),allowing simultaneous de-tection of DHAV-3 and DHAV-1 pathogens.The DAS-ELISA method established in this experi-ment was compared with the RT-PCR method for the detection of 186 clinical samples,and the DAS-ELISA method could simultaneously identify DHAV-1 and DHAV-3,with a compliance rate of 98.9％compared to the RT-PCR method.In conclusion,the established DAS-ELISA method showed good repeatability and high sensitivity,and can be used for the diagnosis of DHAV-1 and DHAV-3,providing technical support for the epidemiological investigation and prevention of Duck Hepatitis A.
