Goat anti human igm hrp

For detection of antibodies against influenza A(H1N1)pdm09 virus in human serum, microtiter plates (Greiner Bio-One) were coated overnight at 4°C with β-PL inactivated virus (250 ng/well). The wells were then blocked with 5% milk, washed thrice and incubated with the diluted sera (1:1600 for IgG; 1:400 for IgM; 1:1600 for IgG1, IgG2, IgG3 and IgG4) for 1 hr at room temperature. To detect the bound IgG antibodies, wells were washed three times with PBS-T and incubated with mouse anti-human IgG, IgG1, IgG2, IgG3 or IgG4 (1:1000 dilution; Sigma Aldrich, USA), followed by rabbit anti-mouse HRP conjugate (1:500 dilution; BioRad) and ABTS. IgM antibodies were detected with goat anti-human IgM HRP (1:250 dilution; Sigma Aldrich, USA). Incubation with class/subclass specific antibodies was for 1 hr at room temperature and that for the conjugate was for 30 min at room temperature; each of the incubation steps, except ABTS, was followed by three washes with PBS-T. The optical density was read at 415 nm.

All sera/plasma was heat-inactivated at 56°C for 30 mins before use in the in-house ELISA. High-binding ELISA plates (Corning, 3690) were coated with antigen (N, S or RBD) at 3 μg/mL (25 μL per well) in PBS, either overnight at 4°C or 2 hr at 37°C. Wells were washed with PBS-T (PBS with 0.05% Tween-20) and then blocked with 100 μL 5% milk in PBS-T for 1 hr at room temperature. Wells were emptied and sera and plasma diluted at 1:50 and 1:25 respectively in milk were added and incubated for 2 hr at room temperature. Control reagents included CR3009 (2 μg/mL), CR3022 (0.2 μg/mL), negative control plasma (1:25 dilution), positive control plasma (1:50) and blank wells. Wells were washed with PBS-T. Secondary antibody was added and incubated for 1 hr at room temperature. IgM was detected using Goat-anti-human-IgM-HRP (1:1,000) (Sigma: A6907) and IgG was detected using Goat-anti-human-Fc-AP (1:1,000) (Jackson: 109-055-043-JIR). Wells were washed with PBS-T and either AP substrate (Sigma) was added and read at 405 nm (AP) or 1-step TMB substrate (Thermo Scientific) was added and quenched with 0.5 M H₂S0₄ before reading at 450 nm (HRP).

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.