As an antigen for the enzyme-linked immunosorbent assay (ELISA), the RBD protein produced in CHO-K1 cells and purified using affinity and ion-exchange chromatography methods (protein purity > 98%) was used [30 (link)]. RBD protein (1 µg/mL in 2 m urea) was adsorbed onto 96-well plates (Greiner Bio-One, Kremsmünster, Austria) at 4 °C overnight, then the plate was washed in PBS with 0.05% Tween 20 (PBST) and blocked with 1% casein solution in the same buffer for 60 min at room temperature. Then, sera were added to the wells in threefold serial dilutions, starting from 1:50, in blocking solution, and incubated for 60 min at room temperature. The plate was washed, and horseradish-peroxidase-conjugated rabbit anti-mouse IgG antibodies (Sigma, St. Louis, MO, USA) were added and incubated for 60 min at room temperature. The substrate 3,3’,5,5’-tetramethylbenzidine (TMB) (Amresco, Dallas, TX, USA) was introduced into the wells of the plate washed with PBST. Optical density was measured at a wavelength of 450 nm using a ChroMate-4300 microplate reader (Awareness Technology Inc., Palm City, FL, USA). Serum dilution was taken as the titer in ELISA, at which the optical density value was more than two times higher than that for the negative control (blocking buffer was added to the wells instead of serum).
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