Mouse anti his mab

To compare the binding affinity of TsCRT and its fragments to C1q, 96-well plates were coated with different amounts of human C1q (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.2, and 1.5 µg/ml) (Millipore, Darmstadt, Germany) in 100 μl/well of coating buffer (100 mM Na₂CO₃/NaHCO₃, pH 9.6) overnight at 4°C. The same amounts of BSA were coated in the control wells. Wells were then washed with PBS + 0.05% Tween-20 (PBST) three times, followed by blocking with 200 µl 3% BSA in PBS at 37°C for 1 h. After further washing, 100 µl of 40 nM rTsCRT, rTsCRT-NP, rTsCRT-P, rTsCRT-S, and rTsCRT-C in binding buffer (100 µl of 20 mM Tris–HCl, pH 7.4, 50 mM NaCl, and 1 mM CaCl₂) was added to each well and incubated for 1 h at room temperature. Mouse anti-His mAb (1:10,000, Tiangen, Beijing, China) and HRP-conjugated goat anti-mouse IgG (1:10,000, BD Biosciences, Franklin Lakes, USA) were used as primary and secondary antibodies, respectively. After washing with PBST, the substrate, o-phenylenediamine dihydrochloride (Sigma, St. Louis, MO, USA) was added; colorimetric detection was performed using TMB and the absorbance measured at 450 nm using an ELISA reader (Thermo Fisher).

Microtiter plates were coated with different amounts of human C1q (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.5 µg) (Merck) in 100 μL/well of carbonate buffer (100 mM Na₂CO₃/NaHCO₃, pH 9.6) overnight at 4°C. Control wells were coated with the same amounts of BSA. Following three washes with PBS + 0.05% Tween-20 (PBST), the wells were blocked with 200 µL of 3% BSA in PBS at 37°C for 2 h. After being washed, 0–1.5 µg of rTs-CRT in 100 µL of 20 mM Tris–HCl, pH 7.4, 50 mM NaCl and 1 mM CaCl₂ were added to each well and incubated for 2 h at 37°C. Then, mouse anti-His mAb (1:10,000, TIANGEN) and HRP-conjugated goat anti-mouse IgG (1:10,000) (BD Biosciences, San Jose, CA, USA) were added and incubated for 1 h at 37°C. After the final washing, the substrate o-phenylendiamine dihydrochloride (OPD, Sigma) was added. The absorbance was measured at 450 nm with an ELISA reader (Thermo).