Hrp conjugated mouse anti flag tag antibody

We purchased 293 FreeStyle (293FS) cells and protein A agarose from Thermo Fisher Scientific. Recombinant human (hHER2), cynomolgus (cHER2), mouse (mHER2), and rat HER2 (rHER2) and other ErbB family member proteins were purchased from Sino Biological (Beijing, China). The pWC1 vector for phage display and bacterial expression and the pDin1 vector used for mammalian expression were designed and generated in our laboratory. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Fc-specific) antibody and HRP-conjugated mouse anti-FLAG tag antibody were products of Sigma (St. Louis, MO, USA). Anti-His-phycoerythrin (PE) conjugate and goat anti-human IgG (Fc-specific)-FITC conjugate were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany) and Sigma (St. Louis, MO, USA), respectively.

A competitive ELISA was used to further analyze the relationship of the binding sites of nCoVmab1 and nCoVmab2. The RBD was coated on the plate overnight at 4 °C. nCoVFab1 and nCoVFab2 at fixed final concentrations of 10 nM and 30 nM, respectively, were mixed with fivefold serially diluted competitive IgGs (nCoVFab1 vs. nCoVmab2 and nCoVFab2 vs. nCoVmab1, at concentrations from 200 to 0.0128 nM) and added to the RBD-coated wells. The bound Fabs were detected by an HRP-conjugated mouse anti-FLAG tag antibody (Sigma-Aldrich). The ELISA procedures were performed as described above.

For ELISA assays, antigen protein was coated on a 96-well plate (Costar) at 50 ng/well in PBS overnight at 4°C. For the soluble VH binding assay, horseradish peroxidase (HRP)-conjugated mouse anti-FLAG tag antibody (A8592, Sigma-Aldrich) was used to detect VH binding. For detection of human Fc protein, HRP-goat anti-human IgG Fc secondary antibody (A18817, Thermo Fisher Scientific) was used. For the competition ELISA, 200 nM of VH-Fc 1-16 was incubated with serially diluted VH 1-16-3 proteins, and the mixtures were added to antigen-coated wells. After washing, competition was detected by HRP-goat anti-human IgG Fc secondary antibody (A18817, Thermo Fisher Scientific). All colors were developed by 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma) and stopped by 1 M H₂SO₄ followed by recording absorbance at 450 nm by iMark™ Microplate Absorbance Reader (Bio-Rad Laboratories). Experiments were performed in duplicate and the error bars denote ± 1 SD.