A New Tool for Complement Research: In vitro Reconstituted Human Classical Complement Pathway

Abstract

The complement, as part of the innate immune system, represents the first line of defense against Gram-negative bacteria invading the bloodstream. The complement system is a zymogen cascade that ultimately assemble into the so-called membrane attack complex (MAC), which lyses Gram-negative bacteria upon insertion into the outer membrane. Traditionally, serum has been used as complement source, for example to study the bactericidal activity of monoclonal antibodies or antibodies raised upon vaccination. Due to the significant donor to donor variability, as well as susceptibility of complement factors to handling and storage conditions, assay reproducibility using human serum is low. Moreover, the presence of pre-existing antibodies and antimicrobial compounds are confounding factors. To remove antibodies from human serum, we applied κ/λ-light chain specific affinity chromatography, however the method severely reduced the complement activity due to the depletion of complement components. Therefore, we attempted to reconstitute human complement—namely the alternative (rAP) and the classical (rCP) pathways—from purified complement factors. We found that adding C1-inhibitor to the mixture was essential to maintain a stable and functional C1 and thus to generate an active rCP. We further confirmed the functionality of the rCP by testing the complement-dependent bactericidal activity of a human monoclonal antibody, A1124 against an E. coli clinical isolate belonging to the ST131 clonal complex, and that of a polyclonal IVIg against a laboratory E. coli strain (MG1655) not expressing LPS O-antigen and capsule. Although the alternative pathway did not have any bactericidal activity by itself, it enhanced MAC deposition induced by rCP and increased the overall bactericidal activity against the ST131 E. coli strain. In conclusion, we report for the first time the successful in vitro reconstitution of the classical pathway of the human complement to establish a serum-free, complement dependent bactericidal assay. This system offers high level of standardization and could support the study of the complement in different research fields.

Complement activation by human IgG antibodies to galactose‐α‐1,3‐galactose

“…However, we refrained from doing this because complement components are likely to be depleted concomitantly, leading to impaired complement activity. 34 In contrast to the conclusion of earlier studies, 4,8,15,[17][18][19] we found that IgG anti-αGal is fully capable of activating the classical complement pathway on pRBCs as well as on bacteria. It is of interest to speculate why other studies observed complement inhibition by IgG anti-αGal.…”

Artificial surface labelling of Escherichia coli with StrepTagII antigen to study how monoclonal antibodies drive complement-mediated killing

“…To circumvent these limitations, we reconstituted the CP with purified complement proteins (Fig. 1 a) by adapting a previously described assay 40 . In this purified CP assay, antibodies and C1-complex are mixed with bacteria for 15 min at 4 °C.…”

“…Purified complement components were used in concentrations in physiological ratio as they occur in (diluted) serum, denoted as percentage serum equivalent 40 , 63 . Per component in 100% serum: C1-complex 135 µg/mL; C1-inhibitor 180 µg/mL; C2 20 µg/mL; C3 1250 µg/mL; C4 400 µg/mL; C5 70 µg/mL; C6 64 µg/mL; C7 56 µg/mL; C8 55 µg/mL; and C9 60 µg/mL.…”

Antibody‐mediated complement activation in pathology and protection

“…For example, there are individual‐ and tissue‐specific differences in the concentration of complement components. To this end, reconstitution of the classical and alternative pathway by a defined mixture of the individual components might be useful in the standardization of complement assays, and may also reduce artifacts originating from other serum components that vary between serum sources 117 …”