Development of an Indirect ELISA Kit for Rapid Detection of Varicella-Zoster Virus Antibody by Glycoprotein E

Abstract

Varicella-zoster virus (VZV), a highly infectious agent that causes varicella (chickenpox), can also cause zoster (shingles), a disorder that is frequently associated with severe neuralgia. A reliable serological VZV diagnostic assay would be useful for identifying unprotected individuals and for surveilling post-vaccination immunoprotection status. Toward this goal, VZV membrane glycoprotein E (gE), the immunodominant VZV protein, served as target antigen in an indirect ELISA kit developed here to detect anti-VZV antibodies in clinical samples. For target antigen preparation, Chinese hamster ovary (CHO) cells were modified to express and secrete the VZV gE ectodomain, which was subsequently purified and used as coating antigen in an indirect ELISA. Ultimately, the optimal purified gE coating antigen concentration was determined to be 2 μg.ml−1 and the OD450nm detection cutoff value was 0.286. The coefficient of variation (CV) of intra-assay and inter-assay were <10 and 15%, respectively. A comparative test of 66 clinical samples showed that the coincidence rate was 93.9% between the indirect ELISA and a commercial varicella-zoster virus IgG ELISA kit. Thus, the indirect ELISA kit developed here may be useful for achieving rapid, sensitive, and specific detection of anti-VZV antibodies.

Development of an indirect ELISA for the identification of African swine fever virus wild-type strains and CD2v-deleted strains

“…To establish a stable CD2v-ex protein expressing CHO-S cell line, CHO-S cells were first transduced with packaged LV suspensions to generate CHO-S cells expressing CD2v-ex (CD2v-ex) and CD2v-ex expression was verified by western blot analysis. A specific diffuse band (70–95 kD) was detected in CHO-CD2v cell culture supernatant ( Figures 1A,B ), consistent with eukaryotic expressed glycosylated proteins' characteristics ( 16 , 20 ), suggesting that CHO-CD2v cells successfully secreted express glycosylated CD2v-ex. Furthermore, the protein was recognized by anti-His-tag antibody and ASFV + serum.…”

“…Supernatant containing the pseudotyped LVs was collected at 72 h post-transfection, centrifuged at 4,000 rpm for 10 min at 4 Establishment of a stable CD v-ex protein expressing cell clone CHO-S cells (Thermo Fisher Scientific,Waltham, MA, USA) were passaged in 125-mL vent-cap cell shaker flasks (NEST, Wuxi, China) containing 25 mL ExpiCHO Expression Medium (Gibco, Grand Island, NY, USA) on an orbital shaker platform (100 rpm). When the cell viability exceeded 95%, 2 × 10 6 cells were mixed with the LV suspension at a ratio of 1:1 (16) in the presence of 5 µg/mL polybrene (Beyotime, Shanghai, China). After 24 h of culture in an orbital shaker at 37 • C, the cells were collected by centrifugation at 500 rpm for 10 min at 25 • C and resuspended in fresh ExpiCHO culture medium containing puromycin (Beyotime).…”