Development and verification of a double-antibody sandwich ELISA for detection of varicella-zoster virus-ORF7 protein
10.13200/j.cnki.cjb.004498
- VernacularTitle:水痘-带状疱疹病毒-ORF7蛋白双抗体夹心ELISA检测方法的建立及验证
- Author:
WANG Menghan
- Publication Type:Journal Article
- Keywords:
Varicella-zoster virus(VZV);
ORF7 protein;
Monoclonal antibodies(mAbs);
Enzyme-linked immunosorbent assay(ELISA)
- From:
Chinese Journal of Biologicals
2025;38(06):731-737+745
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a double-antibody sandwich ELISA for the detection of varicella-zoster virus(VZV)-ORF7 protein,and verify the method for the detection of ORF7 antigen across manufacturing stages of live attenuated varicella vaccine Oka-7S strain.Methods Recombinant plasmid pET-28a-ORF7 was used to induce prokaryotic expression of VZVORF7 protein,which was then purified by Ni~(2+) affinity chromatography column.Using ORF7 prokaryotic protein as the immunogen,the hybridoma cell lines were screened by mouse hybridoma fusion technique,and the monoclonal antibodies(mAbs)were prepared.The antibody titer was detected by indirect ELISA,and the antibody specificity was identified by Western blot.The antigenic epitopes of mAbs were analyzed by ELISA,and the double-antibody sandwich ELISA for VZV-ORF7antigen was developed by antibody pairing screening.The sensitivity,specificity and repeatability of the method were verified,and three batches of VZV Oka-7S harvested samples were detected by using the developed method.Results Four hybridoma cell lines steadily secreting anti-VZV-ORF7 specific antibodies,named M2B5,M2G5,8C2 and 1C4,were produced.The antibody titers of culture supernatant and mouse ascites were 10~4-10~5 and 10~6-10~7,respectively.The purity of purified mAbs was both about 97% in reduced and non-reduced states.All the four mAbs exhibited specific binding to VZV whole virus protein and purified ORF7 prokaryotic protein.M2G5 was selected as the capture antibody and M2B5-HRP as the enzyme-labeled antibody,with the optimum working concentrations of 4 μg/mL and 1:8 000,respectively.The cutoff value was 0.111,and when A_(450)≥0.111,it was determined as positive and <0.111 as negative.The linear range of this method was 3.9-250 ng/mL with a four-parameter fit,and the standard curve equation was y=(0.049 5-2.24)/(1+x/97.8)~(1.27)+2.24,R~2=0.999,with the limit of detection of 2.2 ng/mL.There was no cross-reaction with tetravalent influenza virus split vaccine,mumps virus and measles virus.In addition,the intra-assay and inter-assay CVs of repeatability were all less than 10%.Three batches of VZV Oka-7S strains were all detected to be negative,and the coincidence rate was 100%.Conclusion The developed double-antibody sandwich ELISA against VZV-ORF7 has high sensitivity,good specificity and high repeatability,and can be used for the rapid detection of VZV-ORF7 protein and VZV Oka and Oka-7S strains.
