ELISA for Bacterial Pilus Antibody Detection

The ELISA was performed as described previously [42 (link)]. Briefly, the wells of a 96-well microtiter plate were coated with 50 μl of either a bacterial suspension in PBS (OD₆₀₀ = 0.005) or diluted whole bacterial protein extracts. To prepare whole bacterial protein extracts, one ml of a bacterial suspension at OD₆₀₀ = 0.32 in PBS was mixed with 100 μl of trichloroacetic acid, followed by a 15 min incubation on ice. After a 5 min centrifugation at 20,000× g, the pellet was dissolved in PBS and diluted before being loaded into a 96-well plate. Equal loading was determined using a Bradford total protein assay (Bio-Rad). The samples were allowed to adhere for 2 h and blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature. The samples were incubated for 1 h at 37°C and 5% CO₂ with an anti-FAM20 pilus antibody (1:5,000) [38 (link)] and subsequently incubated with an HRP-conjugated anti-rabbit antibody (1:5,000) for another hour at 37°C. HRP was detected using 3, 3’, 5, 5’-tetramethylbenzidine (TMB), and the reaction was stopped using 1 M HCl. The absorbance at OD₄₅₀ was read using a microplate reader. The experiment was performed twice in triplicate.