Elisa plate reader

The SARS-CoV-2 (Wuhan-Hu-1 strain) S1 protein (Sanyou Biopharmaceuticals Co., Ltd., Shanghai, China) was added at a concentration of 0.1 μg/100 μL/well to 96-well ELISA plates (Corning, NY, USA). Subsequently, serum was added to the wells using 2-fold multiplicative dilution and then incubated at 37 °C for 1 h. Following this, the wells underwent five washes using PBST before 100 μL of horseradish peroxidase-conjugated goat anti-mouse IgG (Thermo Fisher, Waltham, MA, USA) was applied to each well and incubated again at 37 °C for a duration of 40 min. During the serum IgG subtype detection experiments, the antibodies were replaced with horseradish peroxidase-conjugated goat anti-mouse IgG1 and horseradish peroxidase-conjugated goat anti-mouse IgG2a (Abcam, Cambridge, MA, USA). The plate was then washed five times with PBST buffer. Then, 100 μL of TMB substrate (3,3′,5,5′-tetramethylbenzidine) was added for 10 min to allow for color development. Finally, 50 μL of ELISA stop solution (Solarbio, Beijing, China) was added to terminate color development. The results were read using an ELISA plate reader (Gene Company, Hong Kong, China) at a wavelength of 450 nm. The terminal dilution criterion was met when the OD value in the sample wells exceeded 2.1 times that of the blank wells.

An ELISA assay assessing binding antibody levels against the wild-type SARS-CoV-2 was conducted using the wild-type Spike protein. ELISA plates (Corning Costar; Corning, NY, USA) were coated with S protein (Sanyou Biopharmaceuticals Co., Shanghai, China) at a concentration of 5 μg/well and incubated overnight at 4 °C. During the experiment, ELISA plates were blocked with 5% BSA-phosphate-buffered saline (PBS), incubated with serially diluted serum samples, and visualized by reaction with an HRP-conjugated antibody (Abcam, Waltham, MA, USA) and TMB substrate (Solarbio, Beijing, China), using previously described methods [16 (link)]. The absorbance of each well at 450 nm was measured using an ELISA plate reader (Gene Company, Beijing, China). Antibody serum samples yielding OD values at least 2.1-fold higher than the negative control at a test sample dilution of 1:400 were considered positive. Endpoint titers (ETs) were defined as the highest serum dilutions yielding positive OD values. GMTs were calculated as the geometric mean of the ETs of positive serum samples in each group. ELISA assays assessing binding antibody levels against variant strains of SARS-CoV-2 (Alpha, Beta, Gamma, Delta, and Omicron) were conducted using commercial ELISA kits (ACRO Biosystems Co., Beijing, China) according to the manufacturer’s instructions.

VECs transfected with miR-9 mimic or miR-9 control were added to the upper chamber of the Transwell apparatus. After incubation for 12 h, the upper chamber was fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (sigma) for 10 min in the dark and photographed by the phase-contrast microscopy. Finally, the crystal violet was dissolved in 300 μl of 33% acetic acid (sigma) and the absorbance of the solution was measured by an ELISA plate reader (Gene Company Limited, HK, China). To investigate the effect of the miR-9 on the angiogenic activity of VECs in vitro, we performed a tube formation assay. Ninety-six-well culture dishes were coated with 50 μl of matrigel matrix (BD company) and incubated for 30 min at 37 °C. VECs transfected with miR-9 mimic or miR-9 control were seeded onto the solidified gels at a density of 2 × 10⁵ cells/well in 50 μl of culture medium. After incubation for 12 h, the total tube-like structures were photographed by phase-contrast microscopy (× 100) and counted. The above experiments were repeated three times.