A novel method for direct measurement of complement convertases activity in human serum

Abstract

SummaryComplement convertases are enzymatic complexes that play a central role in sustaining and amplification of the complement cascade. Impairment of complement function leads directly or indirectly to pathological conditions, including higher infection rate, kidney diseases, autoimmune-or neurodegenerative diseases and ischaemia-reperfusion injury. An assay for direct measurement of activity of the convertases in patient sera is not available. Existing assays testing convertase function are based on purified complement components and, thus, convertase formation occurs under nonphysiological conditions. We designed a new assay, in which C5 blocking compounds enabled separation of the complement cascade into two phases: the first ending at the stage of C5 convertases and the second ending with membrane attack complex formation. The use of rabbit erythrocytes or antibody-sensitized sheep erythrocytes as the platforms for convertase formation enabled easy readout based on measurement of haemolysis. Thus, properties of patient sera could be studied directly regarding convertase activity and membrane attack complex formation. Another advantage of this assay was the possibility to screen for host factors such as C3 nephritic factor and other anti-complement autoantibodies, or gain-of-function mutations, which prolong the half-life of complement convertases. Herein, we present proof of concept, detailed description and validation of this novel assay.

Mature neutrophils suppress T cell immunity in ovarian cancer microenvironment

“…To evaluate the role of downstream complement proteins in mediating the PMN suppressor phenotype, ascites were pretreated with Ab against C5 or with OmCI, a peptide C5 inhibitor derived from the saliva of Ornithodoros moubata (34,35), prior to coculture. Inhibiting C5, with either antibody or peptide, had a partial abrogating effect on T cell suppression, as compared with Cp40 that fully abrogated the PMN suppressor phenotype ( Figure 3J).…”

Quantitative Mass Spectrometry To Study Inflammatory Cartilage Degradation and Resulting Interactions with the Complement System

“…Wells were blocked with 1% BSA in PBS. Purified complement inhibitors [decay accelerating factor, recombinantly expressed with human Fc tag (21), factor H (FH), purified from plasma as described (22)] or BSA (negative control), fresh culture medium, or medium collected during cartilage culture were mixed with NHS to obtain 50 or 38 mg/ml of control proteins or 50 or 38% of culture media in 0.4 or 4% NHS (for the classical or alternative pathway, respectively). Buffers for dilutions were HEPES ++ (classical pathway) or 40 mM HEPES (pH 7.4), 60 mM NaCl, 10 mM EGTA, 7 mM MgCl 2 (alternative pathway).…”

Antibodies reactive to cleaved sites in complement proteins enable highly specific measurement of soluble markers of complement activation

“…Normal human serum (NHS) was prepared as described (Blom et al, 2014) and heat inactivation was performed by heating at 56°C for 30 minutes. C6 and C9-depleted sera were purchased from Complement Technology (Tyler, TX).…”

“…Hemolytic assay was performed as described previously (Blom et al, 2014). Briefly, sheep erythrocytes were sensitized with anti-sheep IgM (amboceptor), washed in veronal buffer (DGVB ++ ) and incubated with 5% NHS in the same buffer in the presence or absence of C5-blocking antibody.…”