ELISA-based C1q Binding Assay for Recombinant Proteins

An ELISA-based assay was also used to determine the C1q binding activity of TsPmy and its fragments according to the method as previously described [28 (link)]. Briefly, a 96-well ELISA plate was coated with human C1q at 5.0 ug/ml in carbonate buffer (100 mM Na₂CO₃/NaHCO₃, pH 9.6), blocked with 5.0% (w/v) BSA in PBS at 37 °C for 2 h, then incubated with different concentrations of recombinant TsPmy fragments at 25 °C for 1 h. Anti-His mAb at 1:1000 in PBS/1% BSA was used to probe recombinant proteins, and HRP-conjugated goat anti-mouse IgG (BD Biosciences, Franklin Lakes, NJ, USA; 1:5000 in PBS/1% BSA) was used as secondary antibody. For the peptide binding assay, synthesized peptide P2 was labeled with biotin and the C1q coated plate was incubated with different concentrations of biotinylated peptide P2 (B-P2). HRP-conjugated streptavidin (Abcam, Cambridge, UK; 1:10,000 in PBS/1% BSA) served as the detection antibody. After adding the substrate o-phenylendiamine dihydrochloride (OPD, Sigma-Aldrich, St. Louis, MO, USA), absorbance was measured at 450 nm with a plate reader (Thermo Fisher, Life Technologies). BSA coated on the same plate was used as a negative control.