*4.4. ELISA*

IgG specific to SARS-CoV-2 was found using the author's laboratory test system. Polystyrene plates (Nunk) were used for ELISA. The wells of plates were sensitized with both SARS-CoV-2 and SARS-CoV (strain Frankfurt 1, 2002) viral proteins and recombinant antigens at a concentration of 2 μg/mL in 100 μL of 0.05 M sodium phosphate buffer solution (pH 8.0) at 220 ◦C for 18 h. Nonspecific binding sites were saturated with 1% casein solution (Sigma, St. Louis, MO, USA) in TBST (Tris Buffered Saline with Tween containing 0.15 M NaCl; 0.02 M Tris-HCl pH 7.4; 0.05% Tween-20) within 45 min at 37 ◦C. Then, the antigens were incubated with blood sera (with their preliminary depletion of the nonspecific background by 5% of the volume of cell lysates, on which culture the corresponding antigens were produced) with a dilution of 1/100 (double step for titration) in a 0.5% casein solution for 1 h at 37 ◦C. The peroxidase conjugate of anti-species antibodies (Gout anti-human IgG, Sigma) was used at a working dilution of 1/6000 in 0.5% casein solution with incubation for 1 h at 37 ◦C. The immune response was manifested using a liquid substrate based on TMB (3,3 , 5,5 -tetramethylbenzidine). The reaction was blocked by adding 100 μL of 1 N HCl to each well. The absorbency of the substrate– indicator mixture was measured on a Uniscan spectrophotometer at a wavelength of 450 nm. A lysate of uninfected Vero cells and the blood serum of healthy people were used

as a negative control of the antigen. The results of ELISA detected in three repetitions in two independent experiments were calculated using median values.

#### *4.5. Immunoblotting*

The SARS-CoV-2 and SARS-CoV (strain Frankfurt 1, 2002) viral proteins were separated in one wide "pocket" by electrophoresis in a 10% polyacrylamide gel (PAGE) supplemented with sodium dodecyl sulfate (SDS) and transferred to a nitrocellulose membrane (Millipore) in equipment (Cole-Parmer, Vernon Hills, IL, USA) for incubating blotting membranes for 5 h at 50 V, in a 0.025 M Tris-HCl buffer containing 0.192 M glycine (pH 8.3) and 20% ethanol. The sites of nonspecific binding were saturated with a 1% casein solution for 1.5 h at 20–22 ◦C. The whole membrane containing viral proteins was cut into separate strips, then numbered and incubated in separate containers for 4 h at 20–22 ◦C, with human blood sera (dilution 1:100) in a TSBT buffer containing 0.5% casein. After being washed in the TSBT buffer, the membrane strips were treated with anti-species antibodies labeled with horseradish peroxidase (Gout anti-human IgG, Sigma) at a working dilution of 1/6000 in 0.5% casein solution for 2 h at 37 ◦C. Then, the membrane strips were washed with TSBT buffer and developed in a chromagen solution (1 mg/mL 3.3 diaminobenzidine tetrahydrochloride in 50 mM Tris-HCl buffer (pH 7.4) containing 0.145 M NaCl, 20% ethanol, and 0.03% hydrogen peroxide). The reaction was stopped by washing the membrane strips in TSBT buffer. The specific interaction of the antibodies with viral proteins was manifested by the brown color of the stripes.
