**2. Results**

#### *2.1. Patient Characteristics and Sampling*

Overall, 29 people (Supplementary Figure S1) with an average age of 52 (20 to 82 years old) were enrolled and retained as VZV-infected patients, which was based on typical VZV symptoms and using ELISA tests. None of them was (or has been diagnosed) positive for herpes simplex viruses (HSV1/2), underwent an organ transplant surgery, or received an anti-VZV vaccine. Hepatitis A, B, and E were the only pathologies found among the patients. Supplementary Table S1 describes the clinical and epidemiological characteristics of the included patients.

A total of 62 blood samples from the 29 retained VZV-patients and 453 plasmas/sera from random healthy people (used as negative controls) were obtained to test the developed diagnostic approach.

#### *2.2. Patient Samples React with VZV Glycoprotein Depending on Concentration: Cohort Stratification*

As aforementioned, three ELISA kits (ab108781, ab108782, and ab108783 for IgA/IgG/IgM; Abcam) were used to assess the VZV-specific IgA, IgG, and IgM, respectively, in VZVpatient samples from the 29 included VZV-patients. Sixteen samples from random healthy people were used as negative controls. According to the manufacturer's instructions, these ELISA assays were considered standard for VZV-specific antibody detection in patients to confirm and stratify the retained patients as true-positive or equivocal groups. All samples were first serially diluted and assessed for VZV immunoglobulin detection. As a result, all patient samples (but not those from negative controls) reacted with VZV glycoproteins in a concentration-dependent manner (Supplementary Figure S2). These results confirmed the presence of VZV-specific IgA, IgG, and IgM in VZV-patient samples.

Specifically, of the 29 included patients, 21 showed moderate to high reactivity, even at high dilution for IgA, IgG, and IgM detection, respectively. With OD450 ≥ 1 at 1/100 dilution, all these 21 patients were considered true-positive for IgA, IgG, and IgM according to the ELISAs' manufacturer instructions (Figure 1, Supplementary Figures S1 and S2).

**Figure 1.** Results for confirmation and stratification of VZV infection in the recruited cohort. ELISA was performed using ab108781 (**A**), ab108782 (**B**), and ab108783 (**C**) ELISA tests, at 1/100 dilution for each sample. Patients with OD450 above 0.2 (red dotted line) were considered positive; with OD450 between 0.1 and 0.2 (the gray area between positive and negative threshold), they were equivocal; and with OD450 below 0.1 (green dotted line), they were considered negatives.

In contrast, eight patient samples (patient number 6, 11, 13, 15, 18, 20, 21, and 24) showed inconsistent results in IgA, IgG, and IgM detection, respectively. Precisely, each patient sample showed equivocal results (OD450 between 0.1 and 0.21 at 1/100 dilution) for at least one of the three antibody isotypes IgA, IgG, and IgM (Figure 1, Supplementary Figure S2). Therefore, they were considered as equivocal for further analyses.
