A New Tool for Complement Research: In vitro Reconstituted Human Classical Complement Pathway
| Autor: | Gabor Nagy, Eszter Nagy, Michele Mutti, Valéria Szijártó, Katharina Ramoni |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
E. coli ST131
lcsh:Immunologic diseases. Allergy 0301 basic medicine medicine.drug_class Immunology alternative pathway bactericidal activity Monoclonal antibody Microbiology monoclonal Ab 03 medical and health sciences Classical complement pathway 0302 clinical medicine medicine Immunology and Allergy classical pathway biology Chemistry Complement system Complement (complexity) 030104 developmental biology Polyclonal antibodies 030220 oncology & carcinogenesis biology.protein Alternative complement pathway C1-INH Antibody lcsh:RC581-607 Complement membrane attack complex |
| Zdroj: | Frontiers in Immunology, Vol 9 (2018) |
| ISSN: | 1664-3224 |
| DOI: | 10.3389/fimmu.2018.02770 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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