Nunc maxisorp flat bottom 96 wells

Bacterial adhesion to immobilized Fn was evaluated using ELISA. Full-length and Fn fragments were coated onto Nunc Maxisorp flat-bottom 96-wells (468667, Thermo Scientific) using 1 μg, unless mentioned otherwise. Plates were blocked with 2% wt/vol bovine serum albumin in DPBS. Bacteria grown in BaLi medium (1.5 × 10⁸ bacteria, based on OD, see above) were added and incubated for 2 h at 37°C. For competition experiments with immobilized Fn, different concentrations of heparin and bacteria in DPBS were used in Fn-coated wells. For bacterial binding assays to heparin, 0.5, 5, or 50 μg/μL of heparin was used for coating. Interaction of BadA with Fn or heparin was examined by whole-cell ELISA using rabbit anti-B. henselae IgG antibodies followed by HRP conjugated anti-rabbit IgG antibodies in blocking buffer. After each step, three washes were performed using 0.05% vol/vol Tween 20 in DPBS. The assay was developed using TMB solution (T4444, Sigma-Aldrich) and absorbance was spectrophotometrically measured at 450 nm.

The binding of B. henselae to ECM proteins was tested in vitro using an enzyme-linked immunosorbent assays (ELISA). Briefly, 1 μg of human plasma fibronectin (Fn, Sigma-Aldrich) or human collagen-I (Col-I, Merck) was coated o/n at 4°C onto Nunc Maxisorp flat-bottom 96-wells (Thermo Scientific). Wells were blocked with 2% w/v bovine serum albumin (BSA; Sigma-Aldrich) in washing buffer (0.05% v/v Tween 20 in PBS) and incubated for 90 min at 37°C. B. henselae strains grown in BALI medium were resuspended in PBS, added to the wells (ca. 2 × 10⁸ cells), and incubated for 2 h at 37°C. Bound bacteria were detected using anti-B. henselae IgG antibodies (1:1,000 in blocking buffer; Kempf et al., 2000 (link)) and swine anti-rabbit HRP (1:2,000 in blocking buffer). Samples were developed using 3,3′,5,5′-tetramethylbenzidine liquid substrate (TMB; Sigma-Aldrich) for ca. 1 min and the reaction was stopped with 1M HCl. The resulting absorbance was measured at 450 nm using a microplate Sunrise-Basic™ reader (TECAN). All 96-well plates were sealed to prevent evaporation during incubation steps, and were each time immediately followed by three washes in wash buffer. Assays were done in triplicate.