Complement Inhibition by Borrelial Proteins

The inhibitory capacity of borrelial proteins on the CP, LP or the AP was analysed by a microtiter-based approach as described previously^{59 (link)}. Briefly, microtiter plates were coated with either human IgM (30 ng/ml) (Merck, Darmstadt, Germany) for the CP, mannan (1 µg/ml) (Merck, Darmstadt, Germany) for the LP or LPS (100 ng/ml) (Hycult Biotech, Beutelsbach, Germany), for the AP at 4 °C overnight. Following blocking, NHS (1% for the CP, 2% for the LP, and 15% for the AP) pre-incubated with His₆-tagged proteins (10 µg each or increasing concentrations thereof) were added to initiate complement activation. Formation of the MAC was detected by using an anti-C5b-9 antibody (1:500) (Quidel, Athens, USA) and antigen–antibody complexes were visualized by applying HRP-conjugated anti-mouse immunoglobulins (1:1000). The reactions were developed by adding o-phenylenediamine (Merck, Darmstadt, Germany) and measuring the absorbance at 490 nm.