Nunc maxisorp 96 well microtiter plates

Nunc MaxiSorp 96-well microtiter plates (Thermo Fisher Scientific) were coated with 5 μg/ml of His₆-tagged proteins or BSA in PBS at 4°C as described (29 (link)). Wells were washed three times with PBS containing 0.05% (v/v) Tween 20 (PBS-T) and then blocked with Blocking Buffer III BSA (AppliChem, Darmstadt, Germany). Following three washes with PBS-T, 100 μl FH or FHL-1 (5 μg/ml) were added. Following incubation for 1 h at RT, wells were washed thoroughly with PBS-T and binding of complement regulators were then assessed by incubation of the wells with a polyclonal goat anti-FH antiserum (1:1,000). After washing, protein complexes were detected by using HRP-conjugated anti-goat immunoglobulins (1:2,000). Afterwards, o-phenylenediamine (Merck, Darmstadt, Germany) was added to the wells and the absorbance was measured at 490 nm. Additionally, CspA of B. mayonii MN14-1420 was immobilized and incubated with increasing amounts of FH to determine dose-dependency of the binding and to calculate the dissociation constant.
Binding of FH to spirochetes was also assessed by ELISA. Briefly, bacterial cells (2 × 10⁷ cells) in 100 μl PBS were immobilized to Nunc MaxiSorp 96-well microtiter plates at 4°C overnight and binding of FH was detected as described above.

To detect binding of complement components, Nunc MaxiSorp 96-well microtiter plates (Thermo Fisher Scientific) were coated with 100 µl of purified bacterial proteins (5 µg/ml) or BSA (5 µg/ml) in PBS at 4°C overnight as described (31 (link)). Between every incubation step, wells were washed three times with PBS containing 0.05% (v/v) Tween 20 (PBS-T). After blocking with Blocking Buffer III BSA (AppliChem, Darmstadt, Germany) or with PBS containing 0.2% gelatin (w/v) (AppliChem), complement components (10 ng/µl) in PBS were added to the wells. Binding of complement components were then assessed by utilizing specific primary antibodies (dilution 1:1,000). Following incubation for 1 h at RT, HRP-conjugated anti-goat or anti-mouse IgG (dilution 1:1,000) were added and protein complexes were visualized using o-phenylenediamine (Merck). The absorbance was then measured at 490 nm employing PowerWave HT (Bio-Tek Instruments, Winooski, VT, USA).
To determine dose-dependency and to calculate the dissociation constant, CipA was immobilized (5 µg/ml) and incubated with increasing amounts (0 to 50 nM) of C3b, C5, and FI, respectively. The antigen-antibody complexes were detected by using the appropriate antibodies as described above.

Antibodies were tested for complement deposition on S. aureus Newman in an ELISA based assay format. Briefly, S. aureus was grown in RPMI overnight, washed in PBS and adjusted to an OD₆₀₀ of 0.25. Nunc MaxiSorp 96 well microtiter plates (ThermoFisher) were coated overnight with 100 μl of S. aureus culture per well. Plates were fixed with 2% PFA and blocked with 3% BSA in PBS prior to addition of a 1:3 serial dilution of antibody ranging from 0.14 nM – 33.3 nM for 1 h at 25 °C. After washing, 5% protein A/G adsorbed NHS (BioIVT) was added for 1.5 h at 37 °C. Goat anti-C1q antibody (final concentration 1 μg/ml; Genway Biotech, Inc.) was added to detect C1q deposition, followed by donkey anti-goat HRP secondary antibody (1:4000; Jackson ImmunoResearch) and chemiluminescent substrate (ThermoFisher). Luminescence was detected using a SpectraMax i3x plate reader (Molecular Devices). Luminescence values were analyzed by a four-parameter logistic equation over a 7-point response curve (GraphPad Prism) to calculate the binding EC₅₀ of the antibodies. Results are plotted as mean ± standard deviation.