ELISA-Based Binding Assay for Bacterial Interactions

ELISA was performed as previously reported [23 (link)]. In brief, Gram-negative (Vibrio anguillarum, Vibrio harveyi, Pseudomonas fluorescens, Edwardsiella piscicida) and Gram-positive (Streptococcus iniae, Micrococcus luteus, Bacillus subtilis) bacteria were grown as described previously [24 (link)] and resuspended in coating buffer (0.159% Na₂CO₃, 0.293% NaHCO₃, pH 9.6). Lipopolysaccharide (LPS) and peptidoglycan (PGN) (InvivoGen, San Diego, CA, USA) were diluted in coating buffer to a final concentration of 0.1 mg/mL. The bacterial resuspension or the LPS/PGN dilution was added into 96-well microtiter plates, and the plates were placed at 4 °C overnight. After blocking with 5% skim milk for 1 h, the plates were washed three times with PBST. Different concentrations (1, 2, 4, 8, 16, or 32 μg/mL) of AjCASPX2, CARD_AjCASPX2, or Trx were added to the plates. The plates were incubated at 25 °C for 2 h and washed as above. The plates were then incubated with mouse anti-His tag antibody and HRP-conjugated goat anti-mouse IgG successively. The plates were washed five times with PBST, and TMB substrate solution (TIANGEN, Beijing, China) was added to the plates. The plates were analyzed with a microplate reader (BioTek Instruments, Winooski, VT, USA).