Hrp conjugated goat anti mouse igg2a

S1-binding antibodies were detected with SARS-CoV-2 Spike S1 Antibody Titer Assay Kit (Mouse) (Sino Biological Inc., KIT007). RBD-binding antibodies were detected with Spike RBD Antibody Titer Assay Kit (Mouse) (Sino Biological Inc., KIT006) according to the instructions of the manufacturers. Briefly, sera were serially 4-fold diluted in dilution buffer, and 100 μl of the diluted sera was added to spike S1/RBD precoated microplates and incubated for 2 h at room temperature. After the microplates were washed, diluted horseradish peroxidase (HRP)-Rabbit anti-Mouse IgG was added to the plates and incubated for 1 h at room temperature. The plates were then incubated with substrate solution for 20 min. Levels of antibodies were determined by measuring the optical density (OD) at 450 nm with a microplate reader after termination with stop solution. Final end point titers (1/n) were defined as the highest dilution that yielded an absorbance >0.1. For detection of antibody isotypes, HRP-conjugated Goat anti-Mouse IgG1 (Abcam, Cambridge, UK; ab97240) and HRP-conjugated Goat anti-Mouse IgG2a (Abcam, ab97245) were used.

Wells in flat-bottom Maxisorp microplates were coated with 0.15 μg of purified monoclonal antibody 1A4 diluted in 50 mM bicarbonate buffer, pH 9.6, overnight at 4°C. Simultaneously, an additional set of wells were coated with blank buffer to serve as a control with no capture antibody. After three washes in PBS-T, wells were blocked with 1% BSA in PBS-T for 90 minutes at room temperature. Next, a serial titration of SUMO-EhJacob, ranging 16.4 pg to 17.1 ng, was plated and incubated for 60 minutes at room temperature, followed by three washes in PBS-T. Monoclonal antibody 1D3 (1.2 μg/mL) and 125 ng/mL HRP-conjugated goat anti-mouse IgG2a (#ab97245, Abcam, Cambridge, MA) incubated for 1 hour at RT in succession, followed by three and four washes in PBS-T respectively. Finally, wells were developed with 100 μL of 1-Step Ultra TMB solution for 60–80 seconds and stopped with 2 N hydrochloric acid. The microplates were read at 450 nm.