On the day of the testing, the samples were thawed at once and their complement activity was compared with the ELISA based kit as described above

On the day of the testing, the samples were thawed at once and their complement activity was compared with the ELISA based kit as described above. Statistical Analysis Data were analyzed with paired two-tailed < 0.05. Results Removal of Antibodies From Human Serum Impairs the Complement First, we tested the possibility of removing antibodies from normal human serum (NHS) to generate a complement source devoid of pre-existing antibodies for studying complement-mediated bactericidal activity. an active rCP. We further confirmed the functionality of the rCP by screening 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 statement for the first time the successful reconstitution of the classical pathway of the human match to establish a serum-free, match dependent bactericidal assay. This system offers high level of standardization and could support the study of the match in different research fields. Keywords: C1-INH, bactericidal activity, monoclonal Ab, classical pathway, alternate pathway, ST131, MG1655, match system Introduction As part of the innate immunity, the match system is one of the first lines of defense against Gram-negative pathogens. Besides its direct bactericidal activity, the activation of the match system stimulates phagocytosis and triggers pro-inflammatory signaling. The three different pathwaysalternative, classical and lectinconverge into the terminal pathway (TP) that leads to the assembly of the C5b-9 complex, also called Membrane Attack Complex (MAC). The alternative pathway (AP) is usually spontaneously activated around the pathogen surface. The classical (CP) and lectin pathways (LP), are induced by Rabbit polyclonal to annexinA5 an initial receptor-ligand acknowledgement on the target surface. In case of the CP, the antigen-antibody complex (or immune complex, IC) activates the match cascade by binding to C1, a complex created by C1q, C1r, and C1s. In the LP, numerous lectins recognize specific sugar structures around the microbial surface and activate the Mannose Binding Protein-Associated Serine Proteases (MASPs) (1). Both CP and LP are negatively regulated by the C1 inhibitor (C1-INH), which sequesters and inhibits C1r, C1s, and MASPs (2). In the absence of C1-INH, C1 undergoes spontaneous activation (3, 4). Cleavage and activation of the various match factors prospects to the formation of the C5 convertase cleaving C5 to C5a and C5b, initiating the terminal pathway. C5b, attached to the target surface, associates with C6, C7, and C8 and induces the polymerization of C9 monomers, Gallic Acid forming the MAC. MAC is usually a pore forming complex able to place into lipid membranes, including the outer membrane of Gram-negative pathogens to induce membrane damage and cell lysis. The different match pathways interact with each other establishing a complex network in the serum (1, 5, 6). This complexity is further increased by the presence of additional serum bactericidal factors that may take action in concert with the match, or work independently against the invading pathogens (7C9). Therefore, it is hard to dissect the contribution of the different match pathways to the bactericidal action of the serum. It is especially relevant for studies using human serum as match source due to the heterogeneity of the pre-existing antibody repertoires against human pathogens (10) Gallic Acid and match activity (11). While previous studies reported the successful reconstitution of the alternative pathway from individual components (12, 13), up to our knowledge, there have been no reports of a functionally active reconstituted classical pathway, particularly not to study its bactericidal activity. We aimed to establish an model system with purified human match components that allows studying match without confounding factors. Methods and Materials Bacterial Strains and Media strains 81009 (14, 15) and MG1655 (16) were produced in LB or on Trypticase soy agar (TSA) plates (bioMrieux). To prepare overnight cultures, a single colony was inoculated in LB and incubated at 37 C with agitation at 200 RPM overnight. Mid-log-phase bacterial cultures were obtained by diluting the overnight cultures 1:100 in LB and incubating at Gallic Acid 37C at 200 RPM until the cultures reached an OD600 of ~0.5. Match Factors, Sera, and Reagents Rabbit erythrocytes, Gelatin Veronal Buffer with Gallic Acid or without EDTA, as well as the match factors C3 (A113c), H (A137), I (A138), B (A135), P (A139), C5 (A120), C6 (A123), C7 (A124),.