Affinipure goat anti human igg h l

ELISA assays were conducted by pre-coating ELISA plates with RBD (SARS-CoV-2 WT, SARS-CoV-2 BA.1, SARS-CoV-2 BA.2 RBD, Sino Biological) at concentrations of 0.03 μg ml⁻¹ and 1 μg ml⁻¹ in 0.05 M coating buffer (Solarbio, C1055) overnight at 4 °C. The plates were then washed and blocked, after which 100 μl of 1 μg ml⁻¹ antibodies were added to each well and incubated at room temperature for 30 mins. Following incubation, the plates were washed and incubated with 0.25 μg ml⁻¹ Peroxidase-conjugated AffiniPure goat anti-human IgG (H + L) (JACKSON, 109-035-003) for 30 min at room temperature. The reaction was developed using tetramethylbenzidine (TMB) (Solarbio, PR1200), and stopped by adding H₂SO₄. The absorbance was measured at 450 nm using a microplate reader (Thermo Scientific, Multiskan Fc) and the negative control used was the 1 μg ml⁻¹ H7N9 human IgG1 antibody HG1K (Sino Biological, HG1K).

The assay was carried out as described by Zhao RQ, et al [14] . Briefly, wells of 96-well plate were coated with 1.5 μg/mL of SARS-CoV-2 S1-6 × His protein in PBS, blocked with 3% BSA-PBS. Serial dilutions of sera were first diluted with the normal sera from the same species, further diluted with Sample Dilution Buffer (20% Calf serum in PBS, 20%CS-PBS). 100 μL of the diluted samples were transferred to each well of the plates. The captured antibodies were probed with Horseradish-Peroxidase (HRP)-conjugated secondary antibodies. For example, to detect monkey anti-S1 total antibodies, HRP-conjugated AffiniPure Goat Anti-Human IgG (H + L) from Jackson Immuno Research (Catalogue Number: 109–035-003) was used. For IgM detection, HRP-labeled Monoclonal Mouse Anti-Human IgM (μ) from Cellway-lab (Catalogue Number: C030201) was used. After final washes, HRP substrate TMB solution (Beijing Kwinbon, China) was added and absorbencies were measured at 450 nm with a microplate reader.