*4.3. VZV Glycoprotein E Antigen Preparation*

To develop the highly-sensitive diagnostic approach detecting VZV-specific IgA, IgG, and IgM in VZV-patient blood samples, the surface antigen glycoprotein E of VZV (VZVgE) was produced from insect cell cultures using the baculovirus-based vector expression system (BVES) (Invitrogen-ThermoFisher Scientific, Waltham, MA, USA).

#### 4.3.1. Cell Cultures

Two insect cell lines, including *Spodoptera frugiperda* (*Sf9*) and *Trichoplusia ni* (*High Five*, *Hi5*), were used to produce the baculoviruses carrying the gene of interest and express gE protein, respectively. *Sf9* and *Hi5* were cultured at 27 ◦C in SIM-SF and SIM-HF medium (Sino Biological Inc., Beijing, China), respectively, and supplemented with 10% (*v*/*v*) heatinactivated fetal bovine serum (FBS) and 1× penicillin/streptomycin. *Sf9* cell lines were maintained in both adherent and suspension cultures, while *Hi5* cell lines were only cultured in suspension culture. Adherent cell culture was carried out in 6 cm TC plates, and the suspension cultures were maintained in sterile autoclaved Erlenmeyer flasks in a 110 rpm spin shaker (27 ◦C). The suspension *Hi5* cell culture was diluted to 0.7 to 1 million cells every two based on cell viability and density. The adherents *Sf9* cell cultures were detached and diluted every three days based on cell viability and confluence. The cell viability was assessed under a fluorescence microscope using Trypan blue dye (0.4%) and counted using a hemocytometer.
