Horseradish peroxidase hrp conjugated goat anti human igg antibody

Nunc MaxiSorp ELISA plates (Thermo Fisher Scientific) were utilized and coated with 100 μL of recombinant proteins at a concentration of 1 μg ml⁻¹ in a 1× PBS solution. The coating process was performed overnight at 4°C. On the following day, the ELISA plates were washed three times with 1× PBS supplemented with 0.05% Tween 20, and then blocked using 200 μL of 1× PBS with 5% non-fat milk powder for 2 hours at room temperature. After the blocking step, 100 μL of IgGs from the supernatant were added to each well and incubated for 2 hours at 37°C. The ELISA plates were washed three times to remove any unbound IgGs. Next, the ELISA plates were incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody (1:5000, Invitrogen) for 1 hour at 37°C. Subsequently, the ELISA plates were washed five times using PBS containing 0.05% Tween 20. Then, 100 μL of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to each well. After 15 min incubation, 50 μL of 2 M H₂SO₄ solution was added to each well. The absorbance of each well was measured at a wavelength of 450 nm using a Sunrise absorbance microplate reader (BioTek Synergy HTX Multimode Reader).

Recombinant protein of SARS-CoV-2 receptor binding domain (RBD) or S1 protein (Sino biological, Beijing, China) was coated at 2 μg/ml on a 96-well ELISA plate overnight at 4°C. Wells were blocked with 5% non-fat milk in PBST (PBS with 0.05% Tween-20) at 37°C for 30 minutes. Next, the plate was wash with PBST for 3 times. Then, the plate was incubated with 1:100 diluted plasma in PBS at 37°C for 30 minutes. A 1:2500 dilution of horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody (Invitrogen, Carlsbad, CA) or a 1:5000 dilution of Goat-anti-Human IgM HRP (Sigma, St. Louis, MO) was respectively added to the plate and incubate at 37°C for 30 minutes. Wells were wash with PBST for 3 times between each step. At last, TMB (Beyotime, Shanghai, China) was added to the wells and react for 10min. The plates were read at 450nm (Bio-Tek, ELx808). The ELISA test in our study were repeated twice and phosphate buffer saline was used as the negative control of plasma.