ELISA for Anti-CSPG4 mAbs and Serum Antibody Binding

ELISA was used to assess anti-CSPG4 mAbs’ binding to CMPs and to inhibit anti-P10s serum antibodies’ binding back to the P10s peptide by VT68.2 mAb. For binding assays, ELISA plates (Immuno 4 HBX, Thermo Fisher Scientific) were coated overnight with 50 µL per well of 10ug/mL of peptides. After blocking (blocking buffer: PBS with 1% BSA), serially diluted antibodies were added to wells in blocking buffer and incubated at 37 °C for 2 h. Following washing at room temperature, goat anti-mouse IgG secondary antibody (Sigma-Aldrich, Inc., Saint Louis, MO, USA) was added in 1:15,000 dilution in blocking buffer for an hour’s incubation at 37 °C. The inhibition assay was performed similarly, except PADRE-conjugated P10s-immunized human serum [19 (link)] was added in a 1:800 dilution after washing off VT68.2 antibody and binding of the immunized serum antibodies to the peptide were visualized with an HRP-conjugated anti-human IgG (Sigma-Aldrich, Inc.), at a dilution of 1:10,000. Binding was detected by measuring absorbance at 450 nm using the Synergy LX multi-mode reader (BioTek©, Winooski, VT, USA).