Development Of An Enzyme-Linked Immunosorbent Spot Assay-based Neutralization Assay For Varicella-Zoster Virus

VZV, a high contagious virus, causes chicken pox and Herpes zoster (HZ). As better prevention of chickenpox based on live attenuated vOka vaccine, increasing HZ risk of vaccine recipients. By now, HZ caused by VZV is lack of specific teatment. VZV neutralizing antibodies play an important role in blocking of viral diffusion and infection, are also expected to be specific treatment to HZ. The traditional clinical detection methods of VZV cannot meet the needs of fast, accurate and high-throughput at the same time.Therefore, the purpose of this study is to establish a new detection method for VZV neutralization antibodies based on Enzyme-Linked Immunosorbent Spot Assay (ELISPOT).To achieve the goal, the key is obtaining the excellent detection antibody. The antibody to VZV glycoprotein would be the best choice, because the glycoprotein is exposed to the surface of the VZV particles and forming the envelope where the most critical affinity or neutralizing epitope may exist. In this study, the easily accessible cell-free virus with high titer was gained by optimizing the protective solution and the thawing time. Six monoclonal antibodies specifically binding Glycoprotein K (gK) were obtained by mice immunization of cell-free virus. These6monoclonal antibodies’epitopes were gK281-295aa.MAb18A10, one of these six, was identified for its shortest binding epitope and the distribution of key amino acid sites.Once the detail of MAbl8A10was clear, HRP-18A10was prepared for VZV ELISPOT.After further optimization of sensitivity and time-consuming, the VZV Neutralization Enzyme-Linked Immunosorbent Spot Assay (N-ELISPOT) was ultimately established. Compared to the traditional plaque reduction assay, the consistency of neutralization titers gained by the two methods was95.65%, and correct rate of N-ELISPOT detection of mouse serum samples was100%. The N-ELISPOT was reliable. The VZV N-ELISPOT was done in96wells and cost2days. While the plaque-reduction assay was done in24wells or6wells, and cost7 days generally.In summary, this study obtained gK specific antibody18A10and then applied in VZV N-ELISPOT. N-ELISPOT can ensure the correct rate of serum samples with great advantages of time-consuming and throughput compared to traditional plaque-reduction assay. It is expected to supporting clinical serum detection and neutralization epitope research in laboratory by authenticated as one of VZV neutralization assays.