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

• Published in: Frontiers in immunology Vol. 9; p. 2770
• Main Authors: Mutti, Michele, Ramoni, Katharina, Nagy, Gábor, Nagy, Eszter, Szijártó, Valéria
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 04.12.2018
• Subjects: alternative pathway; Animals; Antibodies, Monoclonal - immunology; bactericidal activity; Biological Assay - methods; Blood Bactericidal Activity - immunology; C1-INH; classical pathway; Complement Activation - immunology; Complement Membrane Attack Complex - immunology; Complement Pathway, Alternative - immunology; Complement Pathway, Classical - immunology; Complement System Proteins - immunology; E. coli ST131; Escherichia coli - immunology; Humans; Immunology; monoclonal Ab; O Antigens - immunology; Rabbits; alternative pathway; classical pathway; complement system; C1-INH; E. coli ST131; bactericidal activity; MG1655; monoclonal Ab
• ISSN: 1664-3224; 1664-3224
• DOI: 10.3389/fimmu.2018.02770

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 clinical isolate belonging to the ST131 clonal complex, and that of a polyclonal IVIg against a laboratory 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 strain. In conclusion, we report for the first time the successful 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.