Tmb chromogenic reagent kit

ELISA was used to analyse the interaction of SspA-1, SspA-2 with C3a, C5a protein as described previously [20 (link)]. For C3a and C5a bound assay, 0–7 μg/mL SspA-1T, SspA-2T and BSA as a negative control were immobilized to 96-well plate (Corning, USA), 5 μg/mL C3a and C5a were applied after blocking with 1% BSA in PBST (PBS with 20% Tween-20), respectively. Mouse C3a/C3a des Arg monoclonal antibody (Abcam, England) or rabbit C5a monoclonal antibody (Abcam) was applied at 37 °C for 1 hour to test the direct binding capacity. For competitive binding assay, 5 μg/mL SspA-1T, SspA-2T and BSA were immobilized to 96-well plate, 5 μg/mL C5a combined with 0–20 μg/mL C3a were applied after blocking with 1% BSA in PBST, respectively. Mouse anti-C5 monoclonal antibody (Proteintech, Wuhan, China) was applied at 37 °C for 1 hour to test the C5a binding capacity. The HRP-conjugate goat-anti-rabbit IgG (CST) or HRP-conjugate rabbit-anti-mouse IgG (CST) was used as the second antibody at 37 °C for 1 h before Chromogenic reaction by TMB Chromogenic Reagent kit (Sangon) and detected by Synergy^TM H1 Hybrid Reader (Biotek, USA). These data were presented as means ± standard deviations (SD) from three replicates. The experiments were repeated for three times.