5 5 tetramethylbenzidine tmb

Specific antibody titers of the PoAMA-1-immunized mouse were analyzed using the mouse immune serum by enzyme-linked immunosorbent assay (ELISA). The rPoAMA-1 (1 μg/mL, sodium carbonate buffer) was used to coat 96-well plates and then placed overnight at 4 °C. To reduce nonspecific binding, the wells were blocked with 5% (w/v) non-fat milk in TBST and incubated at 37 °C for 2 h. The plates were washed three to four times with wash buffer (TBST), followed by the addition of serially diluted serum. After 2 h of incubation at 37 °C, the plates were washed with TBST and incubated for 1 h with HRP-conjugated goat anti-mouse antibody (dilution 1: 5000) at RT. Then, the plates were washed again and 3,3′,5,5′-Tetramethylbenzidine (TMB; Beyotime Biotechnology, Beijing, China) was added to the plates at RT for 1 min. The reaction was stopped by adding 50 µL/well 2 M sulfuric acid, and color intensity was determined at 450 nm using microplate reader. All samples were tested in repeats, and the mean absorbance was calculated.

Enzyme-linked immunosorbent assay (ELISA) was used to quantify the SARS-CoV-2 RBD-specific IgG, IgG1 and IgG2a titers in serum based on a previous report [15 (link)], with minor modification. Briefly, ELISA plates were pre-coated with 2 µg/mL RBD of wildtype SARS-CoV-2 overnight at 4 °C. The coated plates were washed with phosphate-buffered saline (PBS) containing 0.05% Tween (PBS-T) and blocked with PBS containing 2.5% bovine serum albumin for 2 h at 37 °C. Mouse sera were serially diluted in PBS-T, followed by incubation of plates at 37 °C for 1 h. After four washes with PBS-T, HRP-conjugated goat anti-mouse IgG (Beyotime, Shanghai, China), goat anti-mouse IgG1 (abcam, Shanghai, China) or goat anti-mouse IgG2a (abcam, Shanghai, China) was applied to plates and incubated at 37 °C for 1 h. After the plates were washed thoroughly, 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) (Beyotime, Shanghai, China) was added into the plates to visualize the reaction. Finally, 1 N H₂SO₄ was added into the plates to stop the reaction, and the absorbance value was collected at 450 nm using a Tecan Infinite M200PRO microplate reader (Männedorf, Switzerland). The endpoint titers were expressed as the highest reciprocal serum dilution exhibiting an absorbance of 450 nm > 2.1-fold over the background values.