Bead arrays for antibody and complement profiling reveal joint contribution of antibody isotypes to C3 deposition

The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen β and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen β peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement activation plays role in the pathomechanism

Complement C4 monitoring in the follow-up of chronic hepatitis C treatment

The overall role of complement in the host–pathogen relationship is now well understood. However, its involvement at a chronic stage of infection, such as chronic hepatitis C, is less well documented. Here, results are reported which point to the use of specific C4 monitoring in the follow-up of HCV patients. This study concerns 66 patients with chronic HCV infection, treated with interferon alpha 2b alone or with interferon alpha 2b + ribavirin, and 50 healthy adults as controls. Complement blood tests were performed to measure C1q, C3, C4, mannan binding lectin (MBL), C1s-C1 inhibitor complexes, total (CH50) and C4 (C4H) haemolytic activity; specific C4 activity was taken as the C4H/C4 protein ratio. Rheumatoid factor (RF) levels were also measured. A significant reduction in CH50 and specific C4 activity in HCV patients, compared with the healthy controls, was observed before the onset of treatment; the other parameters were not affected and no C1s-C1 inhibitor complexes were detected. At the same time, a significant reduction in specific C4 activity was observed in relapsers compared with sustained responders. These results point to a potential predictive function of C4 specific activity to monitor the response to therapy. Restoration of specific C4 activity at 6 months was better in responders than in non-responders. Complement activation in chronic hepatitis C does not seem to involve the C1 stage of the classical pathway. A negative correlation between specific C4 activity and RF titres suggests a possible involvement of RF in C4 activation, via the lectin pathway. Specific C4 monitoring appears to be a valuable tool for the follow-up of chronic hepatitis C treatment, together with the other conventional investigations