4.3.2. Molecular Cloning, Expression, and Purification of VZV-gE Protein

Briefly, the sequence of the mature extracellular region of VZV-gE (GenBank Accession number MH709377.1) was retrieved by PCR from the General Biosystem company's synthetic construct using the following forward and reverse primers: 5 -ATTTCCAAGGTTCTT CCGTCTTGCGATACGATGATTTTCACATC-3 and 5 -GACAAGCTTGGTACTTAATATCG TAGAAGTGGTGACGTTCCGGG-3 , respectively. In the meantime, the His-tag-modified transfer vector (pI-SUMO-Star-His) was linearized using primers (forward: 5 CTTCTACGA TATTAAGTACCAAGCTTGTCGAGAAGTACTAGAGG3 and reverse: 5 TATCGCAAGAC GGAAGAACCTTGGAAATAAAGATTCTCGCTGCC3 ) containing sequences that overlap the VZV-gE 5 and 3 end sequences. The linear fragments were ligated following the Gibson Assembly method's instructions. The successful construct was transposed into bacmid using *DH10Bac E. coli* strain and purified for transfecting *Spodoptera frugiperda (Sf9)* insect cell lines, which produced recombinant VZV-gE baculoviruses. Expanded recombinant baculovirus stock was used to infect two million *Trichoplusia ni (High Five, Hi5)* insect cell lines for expressing the recombinant VZV-gE protein. The protein was harvested three days post-infection by high-speed centrifugation and purified from the supernatant.

A couple of purifications steps, including membrane diafiltration (Vivaflow 200), dialysis, ion-nickel column purification, size-exclusion chromatographic purification, and ultra-centrifugation were conducted to purify the protein. The purified protein (Supplementary Figure S3) was stored in HEPES buffer saline (HBS: 20 mM HEPES, 250 mM NaCl), an amine-free buffer, which is required for the further experiments.

#### *4.4. Preparation and Validation of the CLIA-Based Diagnostic Kit*

The highly purified VZV-gE protein was employed to make the CLIA-based diagnostic kit. The purified VZV-gE protein was first biotinylated using EZ-Link Sulfo-NHS-LC-LC-Biotin, No-Weigh™ Format kit (Thermo Fisher, n◦A35358, Waltham, MA, USA) following the manufacturer's instructions. Then, the biotinylated protein was immobilized onto magnetic beads using an Invitrogen Dynabeads™ MyOne™ Streptavidin C1 kit (Thermo Fisher), following the manufacturer's instructions, and further blocked (or saturated) with 2% of bovine serum albumin (BSA) to avoid non-specific interactions or background. Immobilizing the antigen protein onto a solid phase (here beads) is necessary for immunoblotting IgA, IgG, and IgM antibodies on a solid phase. The detection procedure below was performed with a fully automatic chemical luminescent immune analyzer, Kaeser 1000 (Kangrun Biotech, Guangzhou, China). Secondary antibodies anti-human IgA, IgG, or IgM conjugated with acridinium were used to detect the caught VZV-gE specific IgG, IgA, or IgM antibodies, respectively. The detected chemiluminescent signal over the background signal was automatically obtained as relative light units (RLU).

These collections, which contain all the described buffers and components for CLIA of VZV gE-specific IgA, IgG, and IgM, are referred to as VZV-gE-IgA, VZV-IgG, and VZV-gE-IgM kits here. As described above, each diagnostic kit was developed independently, with the corresponding secondary antibody conjugated with acridinium.

A first test batch of a two-fold serial dilution of the ELISA-based true-positive and healthy plasmas/sera was conducted to assess the reliability of the antibody detection kit regarding sample dilution. A subsequent CLIA test was performed to determine the diagnostic kit performances.

#### *4.5. Statistical Analysis*

ELISA tests were triplicate, and the results were transformed, fitted, and presented as mean ± SD. To determine the optimal cut-off values (criteria) and evaluate the diagnostic characteristics of VZV-gE-IgA, IgG, and IgM kits, receiver-operating characteristic (ROC) analyses were performed using MedCalc software. Thus, the specificity and sensitivity of the gE-specific IgA, IgG, and IgM detection kits were determined according to the following formulas:


A Mann–Whitney test was used to assess any significant variation of VZV gE-specific IgA, IgG, or IgM level between equivocal and true-positive ELISA-based categories. The same analysis was used to assess any significant correlation of the antibody levels regarding the age ranges. An analysis of variance (ANOVA) test was conducted using the Kruskal–Wallis approach to determine any difference of antibody level between the three independent groups, including positive, equivocal, and negative. A p-value less than 0.05 defined a hypothesis as statistically significant. All the above analyses were integrated into GraphPad Prism5.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/pathogens11010066/s1. Supplementary Figure S1: Flow chart of patient inclusion and analyses, Supplementary Table S1: Epidemiological and clinical patient data, Supplementary Figure S2: ELISA results of all the 29 included patients, Supplementary Figure S3: Purification of VZV-gE recombinant protein using baculovirus-based vector expression system (BVES), Supplementary Figure S4: Validation of VZV-gE specific IgA, IgG, and IgM detection based on CLIA approach.

**Author Contributions:** Conceptualization, A.J.K.K. and T.J.; data curation, A.J.K.K.; formal analysis, A.J.K.K.; funding acquisition, T.J.; investigation, S.Z.; methodology, A.J.K.K.; project administration, S.Z. and T.J.; resources, A.J.K.K. and T.J.; software, A.J.K.K.; supervision, S.Z. and T.J.; validation, A.J.K.K., S.Z. and T.J.; writing—original draft, A.J.K.K.; writing—review and editing, A.J.K.K., S.Z., J.X., A.Z., H.M., G.X., Y.D., F.A.N.B., A.M., Z.D., Y.Y., C.F., W.Z., R.C., K.Z. and T.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB29030104), the National Natural Science Fund (Grant No.: 31870731 and 31971129), the Fundamental Research Funds for the Central Universities, Jack Ma Foundation, and the 100 Talents Program of The Chinese Academy of Sciences, the Chinese Government scholarship program, and the CAS-TWAS scholarship program.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China (USTC), n◦ 2021-ky269.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patients to publish this paper.

**Data Availability Statement:** Restrictions apply to the availability of the patients data. Patient data was obtained from the First Affiliated Hospital of the University of Science and Technology of China (USTC) and are available from the corresponding authors with the permission of the First Affiliated Hospital of the University of Science and Technology of China (USTC), while patient sample-related experimental research data was obtained from our Laboratory.

**Acknowledgments:** We would like to thank the staff and patients at the Department of Dermatology and the Anhui Provincial Hospital, The First Affiliated Hospital of USTC, for their support in providing samples and collecting clinical data.

**Conflicts of Interest:** Tengchuan Jin, Arnaud John KOMBE KOMBE, and Huan Ma in USTC have jointly applied for a joint patent related to the antibody detecting kits. Other authors declare no conflicts of interest.
