Blocking buffer 3 bsa

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.

Microtiter plates were coated with His-tagged proteins (5 µg/ml each) over night at 4 °C. After blocking with Blocking buffer III BSA (AppliChem, Darmstadt, Germany) for 2 h at room temperature, 5 μg/ml of complement components C3, C3b, C4, C4b, C5 or complement regulators FH or C4BP in PBS was added and incubated for 1 h at room temperature. Bound proteins were detected with the appropriate primary antibody and protein complexes were identified using secondary horseradish peroxidase-coupled antisera. The reaction was visualized with 1,2-phenylenediamine dihydrochloride (Sigma-Aldrich, Taufkirchen, Germany) and absorbance was measured at 490 nm.
For competitive ELISA, microtiter plates were coated with His-tagged borrelial proteins as described above. After blocking, complement components FH and C4b as well as FH and C4BP, (5 µg/ml each) in 100 µl PBS were added and incubated for 1 h at room temperature. Bound protein complexes were detected using anti-C4 or anti-FH antibodies and antigen-antibody complexes were detected as described above.
For dose-dependent binding, microtiter plates were coated with 100 ng of CbiA and after blocking increasing concentrations (5, 10, 15, 20, and 25 nM) of complement components were added and antigen-antibody complexes were 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.