*2.3. Anti-VZV-gE IgA, IgG, and IgM Detection-Based VZV Diagnostic Kit Has High Sensitivity/Accuracy*

The highly purified VZV-gE antigen (Supplementary Figure S3) was used to make the CLIA-based IgG, IgA, and IgM detection kit, respectively. Then, this developed approach was tested for detecting VZV-gE-specific IgA, IgG, and IgM in sera and plasmas of patients. The reliability of the developed CLIA diagnostic kit was assessed with a two-fold serial dilution of the 62 samples from the 29 patients along with the 16 negative samples used in standard ELISA. As expecting and similar to the standard ELISA results, the CLIA approach showed sample dilution-dependent results (Supplementary Figure S4). These results validated the CLIA diagnostic approach.

Then, to assess the performance of the CLIA approach, the cohort of 42 samples from the 21 true-positive patients and 453 independent samples (plasma or serum) from healthy people were tested by the developed VZV-gE-IgA/IgG/IgM kit. Before testing, samples were pre-treated (virus-inactivated) and diluted 20 times with dilution buffer (PBS) supplemented with 2% BSA. ROC analysis results showed sensitivities of 95.2% (IC95%: 76.2–99.9%), 95.2% (IC95%: 83.8–99.4%), and 97.6% (IC95%: 87.4–99.9%) and specificities of 98.0% (IC95%: 96.3–99.1%), 100% (IC95%: 99.2–100%), and 98.90% (IC95%: 97.4–99.6%) for IgA, IgG, and IgM detection, respectively (Figure 2A–C, Table 1). The cut-offs (criterion) for the IgA, IgG, and IgM diagnostic tests were >78662 RLU, >23450 RLU, and >89634 RLU, respectively (Figure 2A–C).

**Figure 2.** Performance of VZV-gE specific IgA, IgG, and IgM detection kits. The receiver-operating characteristic (ROC) curve analysis for detection of anti-IgA, IgG, and IgM antibodies against VZV-gE protein obtained from 42 ELISA positive samples, regardless (**A**–**C**), and considering as positive (**D**–**F**) the equivocal patient samples. The area under the curve (AUC) and the p-value are shown.

**Table 1.** Sensitivity, specificity, and overall agreements of each VZV-gE-specific IgA, IgG, and IgM kit and their combinations in diagnosing varicella–zoster.



**Table 1.** *Cont.*

CLIA-based kit diagnoses features obtained regardless # and regarding as positive \* the equivocal samples; NA: non-applicable.
