Hrp conjugated mouse anti his monoclonal antibody

The evaluations of RBD-CD protein levels in HEK-293 cell culture supernatant were performed by ELISA. High binding Costar 96-well plates (Sigma-Aldrich, St. Louis, MI, USA) were coated with 5 μg/mL of hFc-ACE2 in coating buffer (0.1 M Na₂CO₃/NaHCO₃, pH 9.6). The plates were incubated at 4 °C for 16 h and then they were washed three times with washing buffer (PBS + 0.05% Tween 20). Blocking was performed for 1 h at 37 °C with blocking solution (3% skim milk in PBS + 0.05% Tween 20). The samples were applied to the plates and incubated at 37 °C for 2 h. Then, the plates were washed three times with washing buffer. Afterward, a horseradish peroxidase (HRP)-conjugated mouse anti-His monoclonal antibody (1:20,000, Sigma-Aldrich) diluted in blocking solution was added and the plates were incubated at 37 °C for 30 min. The plates were washed again and a mix of 3,3–5,5-tetramethylbenzidine (TMB) and H₂O₂ diluted in citrate-phosphate buffer (pH 4.2) was added as substrate. The reaction was stopped with 2M H₂SO_4. Optical density was measured at 450 nm on a microplate reader.

After protein electrophoresis, proteins were transferred to a nitrocellulose membrane (GE Healthcare). Membranes were blocked overnight at 4 °C with 5% skim milk in washing buffer (PBS pH 7.5, Tween-20 0.05%). After the blocking step, the membrane was washed three times for 5 min with washing buffer.
The membrane was incubated for 1 h at room temperature in shaken conditions with a horse-radish peroxidase (HRP)-conjugated mouse anti-His monoclonal antibody (1:2000, Sigma-Aldrich) diluted in PBS. Then, the membrane was washed again and the reaction developed.
Sera from convalescent patients were also used. All individuals gave their written informed consent for the use of their serum. The previously blocked membrane was incubated for 2 h at room temperature in shaken conditions with sera from convalescent patients (1:250). Then, the membrane was washed again and incubated with HRP-conjugated anti-human IgG (1:10,000, Jackson InmunoResearch) diluted in blocking buffer. After a step of incubation for 1 h at room temperature in shaken conditions, the membrane was washed again and the reaction developed.
For visualizing the proteins of interest previously marked by the HRP-conjugated antibodies, two different substrates were used: ECL detection system (Amersham) and 3,3′ diaminobenzidine (DAB).