Figure 1. Complement activation and adherence of complement proteins to Borrelia spirochetes incubated in hirudin plasma.

A) The ability of the Borrelia strains B. afzelii K78 and B.gariini LU59, resistant and sensitive to complement activation, respectively, to activate complement in hirudin plasma (n = 8) was assessed by analyzing the generation of C3a and sC5b-9 using ELISA. Data are normalized against plasma incubated without bacteria and are presented as means ± SEM. B) Borrelia were incubated with hirudin plasma (n = 6), and bound proteins were analyzed using an in-house-developed particle ELISA. Data are shown as absorbance values of bacteria incubated in plasma, normalized against bacteria incubated in PBSCa²⁺, and are presented as means ± SEM. C) Quantification of bound C3-fragments (using the β-chain that is not proteolytically cleaved) and FH. Results are presented normalized to either K78 or LU59 as mean of six Western blots. D) Silver stained SDS–PAGE of B. afzelii K78 and B.gariini LU59 non-opsonized (PBS) or opsonised (Pl). In addition, C3 and its fragments (E) and FH (F) bound to bacteria incubated with plasma (Pl) were visualized using Western blot with bacteria incubated in PBSCa²⁺ as controls, and purified C3, C3b and iC3b or FH as references. Insert: loading control, consisting of a 20kDa Borrelia protein that was detected on the membranes after detection with antibodies.