Indirect ELISA for SARS-CoV-2 RBD Antibodies

To examine SARS-CoV-2 RBD-specific antibodies in the mouse sera, indirect ELISAs were conducted as published before [9 (link)]. Briefly, plates were coated with 0.2 µg/well of SARS-CoV-2 RBD proteins from different variants. Mouse sera samples were 3-fold diluted from 1:200 to 1: 11,809,800 in 0.1% BSA in an assay buffer (0.05% Tween20 in 1x PBS). Samples were prepared and measured in duplicate. Assay controls included a 1:400 dilution of pooled naïve mouse sera (negative control), 1:10,000 dilution of pooled high titer mouse (positive control), and assay buffer as blanks. A total of 100 µL/well of 1:6000 goat anti-mouse IgG HRP in assay buffer was added. After incubation, plates were washed five times, followed by adding 100 µL/well of TMB substrate. Plates were incubated for 15 min at room temperature (RT) while protected from light. After incubation, the reaction was stopped by adding 100 µL/well of 1 M of HCl. The absorbance at a wavelength of 450 nm was measured using a BioTek Epoch 2 spectrophotometer. For each sample, the titer was determined using a four-parameter logistic regression curve of the absorbance values. The titer cutoff value: negative serum control + 3 x standard deviation of the negative serum control.