Bacterial Binding Assay by ELISA

Protein binding to the bacteria and bacterial components was determined by ELISA as described previously [21 (link)]. Briefly, bacteria (10⁸ CFU/mL) or bacterial cell wall components (PGN, LTA, or LPS, 200 μg/mL) were each added to a 96-well microtiter plate. The plate was incubated at 4 °C for 12–16 h, followed by washing in PBST (PBS with 0.05% Tween 20) three times. Two hundred microliters of 5% skim milk (Solarbio, Beijing, China) dissolved in PBST was added to the plate. After incubation at 37 °C for 2 h, the plate was washed as above. Two hundred microliters of Crus2, Crus2DC, or PBS (control) was added to the plate. After incubation at 37 °C for 1 h, the plate was washed as above. The bound protein was detected by adding anti-His antibody conjugated with HRP (1/1000 dilution) (ABclonal, Hubei, China) into the plate. After incubation at 37 °C for 1 h, the plate was washed five times with PBST. The TMB substrate solution (Tiangen, Beijing, China) was added to the plate for color development. ELISA stop solution was then added to the plate. The absorbance at 450 nm was measured using a multifunctional microplate reader. The binding index = OD₄₅₀ of protein/OD₄₅₀ of PBS.