Aquacide 2

For PRNT₅₀, one day before infection, 1 × 10⁵ Vero cells/ml were seeded in 24‐well plate using MEM containing 10% FBS and antibiotics. 1:5 diluted serum samples were heat‐inactivated for 30 min at 56°C. Four‐fold serial dilution was performed and mixed with an equal volume of 20‐40 pfu of SARS CoV‐2. The serum‐virus mixture was incubated for 1 h at 37°C in a humidified incubator with 5% CO₂. After incubation, 100 µL of the mixture was added in duplicate to 24‐well plate and incubated for 1 h at 37°C in humidified incubator with 5% CO₂. After incubation, 1 ml of 1% overlay media containing MEM, Aquacide‐II (Merck), 2% FBS and antibiotics was added to the Vero cell monolayer. Plates were incubated for 5 days at 37°C in a humidified incubator with 5% CO₂. Five days postinfection, overlay media was discarded, cells were fixed using 3.7% formaldehyde and after washing with PBS, cells were stained using 1% crystal violet. Plates were washed and kept for air dry. Plaques were counted manually and PRNT₅₀ titer was determined using Karber's formula as described earlier.¹⁴ Samples with PRNT₅₀ titer ≥20 were considered seropositive.

Maltooligosaccharide (G_1-4), soluble potato starch, corn starch, bovine serum albumin and ampicillin sodium salt were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tryptone and yeast extract were obtained from Difco (Bacton Dickinson and company, Sparks, MD, USA). β-CD was purchased from AppliChem (Darmstadt, Germany). Standard maltitol and D-sorbitol were obtained from TCI (Toshima, Kita-ku, Tokyo, Japan). Aquacide II, methanol, isopropanol, ethyl acetate, acetonitrile, HPLC-grade water, potassium dihydrogen phosphate, dipotassium phosphate and TLC silica gel 60 F254 glass plates (20 cm in height) were purchased from Merck (Darmstadt, Germany). IPTG, glycine and Tris-HCl were obtained from Vivantis (Sendirian Berhad, Shah Alam, Malaysia). Sulfuric acid was obtained from Carlo Erba Reagents (Val de Reuil, France). The commercial glucose oxidase kit was obtained from Human mit-diagnostics GmbH (Idstein, Germany). Other chemicals used were of an analytical grade.

For PRNT, 1 day before infection, 1 × 10⁵ cells/mL were seeded in a 24-well plate using MEM containing 10% FBS and antibiotics. Serum samples diluted at a ratio of 1:5 were heat inactivated for 30 minutes at 56°C. Four-fold serial dilution was performed and mixed with equal volume of 40 to 80 pfu of virus. The serum–virus mixture was incubated for 1 hr at 37°C in humidified incubator with 5% CO₂. After incubation, 100 μL of the mixture was added in duplicate to 24-well plate and incubated for 1 hour at 37°C in humidified incubator with 5% CO₂. After incubation, 1 mL of 1% overlay media containing MEM, Aquacide-II (Merck, Darmstadt, Germany), 2% FBS and antibiotics was added to Vero cell monolayer. Plates were incubated for 5 days at 37°C in humidified incubator with 5% CO₂. At 5 days postinfection, overlay medium was discarded, cells were fixed using 3.7% formaldehyde, and after washing with phosphate-buffered saline, cells were stained using 1% crystal violet. Plates were washed and air-dried. Plaques were counted manually, and PRNT₅₀ titer was calculated using Karber’s formula, log10 PRNT₅₀ = m − Δ (Σp − 0.5), where m is the log10 of the highest dilution and Δ is the constant interval between dilutions expressed as log10, as previously described.¹⁹ (link)