1 2 phenylenediamine dihydrochloride

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.

Sodium carbonate, Folin-Ciocalteu phenol reagent, sodium acetate, citric acid, potassium chloride, hydrochloric acid, iron(III) chloride hexahydrate, 2,4,6-tripyridyl-S-triazine (TPTZ), 1,2-phenylenediamine dihydrochloride (OPDA), all polyphenolic (caffeic acid, chlorogenic acid, coumaric acid, ferulic acid, hyperoside, isoquercitrin, quercetin, quercitrin, rutin, ellagic acid, gallic acid, (+)catechin, (−)epicatechin, castalagin, and vescalagin) and terpenic standards (limonene, phellandrene, sabinene, γ-terpinene, and terpinolene), potassium dihydrogen phosphate, phosphoric acid and HPLC-grade methanol and acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethylenediaminetetraacetic acid disodium salt was purchased from AMRESCO (Solon, OH, USA). Sodium fluoride was purchased from Riedel-de Haen (Seelze, Germany). Milli-Q ultrapure water was produced by Sartorius Stedim Biotech mod. Arium (Sartorius, Göttingen, Germany). Cetyltrimethylammonium bromide (cetrimide), ascorbic acid (AA) and dehydroascorbic acid (DHAA) were purchased from Extrasynthése (Genay, France). Acetic acid, ethanol, organic acids (citric acid, malic acid, oxalic acid, quinic acid, succinic acid, and tartaric acid) and HPLC-grade formic acid were purchased from Fluka BioChemika, Buchs, Switzerland.