Preparation of monoclonal antibodies against VP6 protein of human group A rotavirus and development and verification of double-antibody sandwich ELISA for antigen detection
10.13200/j.cnki.cjb.004442
- VernacularTitle:人A组轮状病毒VP6单克隆抗体的制备及双抗体夹心ELISA检测方法的建立和验证
- Author:
ZHAO Ying
- Publication Type:Journal Article
- Keywords:
Human group A rotavirus(RVA);
VP6;
Eukaryotic expression;
Monoclonal antibody;
Double antibody sandwich ELISA
- From:
Chinese Journal of Biologicals
2025;38(03):330-337+343
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare monoclonal antibodies against VP6 protein of human group A rotavirus(RVA),develop a double-antibody sandwich ELISA detection method,and to verify and preliminarily apply the method,in order to provide more choices for the rapid diagnosis,epidemiological investigation and virus surveillance of RVA in China.Methods VP6protein of human RVA G9P [8] strain was expressed and purified with baculovirus expression system,and then applied to immunize five female BALB/c mice to generate monoclonal antibodies.The median effective concentration(EC_(50)) of the monoclonal antibodies was determined by indirect ELISA,and the binding activity of monoclonal antibodies to RVA was determined by Western blot and indirect immunofluorescence assay(IFA).Using monoclonal antibody pairing,one strain was used as coating antibody and the other strain was labeled with HRP for detection,and a double-antibody sandwich ELISA was developed.The sensitivity,specificity and precision of the method were further verified.RV clinical stool samples were taken and detected by Oxoid ProSpecT commercial kit and the developed double-antibody sandwich ELISA respectively,and the coincidence rate of the detection results was compared.Results The purified soluble VP6 protein was obtained,with a purity of over 90% and an expression level of about 4.5 mg/L.Two strains of RVA VP6 monoclonal antibodies were screened,named 1L3 and 4F22,and the EC_(50) of them binding to VP6 protein was 1.777 and 3.748 ng/mL,respectively,indicating that the two strains of monoclonal antibodies could bind well to RVA.The sandwich ELISA method for detection of human RVA was developed using 1L3 as the capture antibody and HRP labeled 4F22 as the detection antibody.The minimum detectable amount of RVA VP6 protein was 156.25 ng/mL.There was no cross-reaction between the two monoclonal antibodies and human Norovirus(NoV),Sapovirus(SaV) and adenovirus(AdV).The coefficients of variation(CVs) of intra-batch and inter-batch precision verification were both less than 10%.The 192 clinical stool samples of RV were detected by using this method,and compared with the commercial kit results,the positive coincidence rate and negative coincidence rate were 99% and 98%,respectively.Conclusion The double-antibody sandwich ELISA against human RVA developed in this study has good specificity,sensitivity,and precision,which can be used for the detection of human RVA.
