On-chip Complement Activation Adds an Extra Dimension to Antigen Microarrays

Abstract

Antibody profiling on antigen microarrays helps us in understanding the complexity of responses of the adaptive immune system. The technique, however, neglects another, evolutionarily more ancient apparatus, the complement system, which is capable of both recognizing and eliminating antigen and serves to provide innate defense for the organism while cooperating with antibodies on multiple levels. Complement components interact with both foreign substances and self molecules, including antibodies, and initiate a cascade of proteolytic cleavages that lead to the covalent attachment of complement components to molecules in nanometer proximity. By refining the conditions of antibody profiling on antigen arrays we made use of this molecular tagging to identify antigens that activate the complement system. Antigen arrays were incubated with serum under conditions that favor complement activation, and the deposited complement C3 fragments were detected by fluorescently labeled antibodies. We used genetically C3-deficient mice or inhibition of the complement cascade to prove that the technique requires complement activation for the binding of C3 to features of the array. We demonstrate that antigens on the array can initiate complement activation both by antibody-dependent or -independent ways. Using twocolor detection, antibody and complement binding to the relevant spots was measured simultaneously. The effect of adjuvants on the quality of the immune response and binding of autoantibodies to DNA with concomitant complement activation in the serum of mice suffering from systemic autoimmune disease was readily measurable by this new method. We propose that measurement of complement deposition on antigen microarrays supplements information from antibody binding measurements and provides an extra, immune function-related fingerprint of the tested serum. Molecular & Cellular Proteomics 6: 133-140, 2007.

Anti-factor B autoantibody in dense deposit disease

“…Since factor B:IgG complexes were detected in the patient, and the autoantibodies were of IgG3 isotype, we investigated the possibility of immune complex-mediated classical pathway activation by analyzing the complement activatory capacity of the autoantibodies on antigen microarray (Papp et al, 2007). The observed, somewhat elevated C3 deposition on factor B spots from the patient's plasma are unlikely to be of major pathological significance, because neither factor B nor IgG are detected in glomerular deposits in DDD (Sethi et al, 2009).…”

“…In addition, because IgG3 antibodies are known as a complement activating IgG isotype, we wanted to examine the possibility that the anti-factor B autoantibody plays a pathogenic role in the patient by activating the classical pathway. A protein microarray was used to characterize the functional autoantibody profile of the patient (Papp et al, 2007). In this assay antigens are exposed to whole serum, thus this approach better mimics the in vivo situation for the formation of antigen-antibody complexes than do purified antibodies in ELISA.…”

Detection of Complement Activation on Antigen Microarrays Generates Functional Antibody Profiles and Helps Characterization of Disease-Associated Changes of the Antibody Repertoire

“…Interestingly, isotype differences in the recognition of selfAgs within the same serum sample have been already shown (10). To circumvent the problem of detailed isotype, glycoform, and affinity determinations of Abs we have introduced the measurement of deposited complement component C3 fragments on Ag microarrays (11).…”

Characterization of factors influencing on-chip complement activation to optimize parallel measurement of antibody and complement proteins on antigen microarrays

“…Parallel measurement of these two reactions provides a functional map of the "immunome". On-chip complement activation is suitable for the parallel measurement of bound antibody and antigen specific complement activation (Papp et al, 2007). Importantly, reproducible and comparable measurement of complement activation requires controlled sample collection, storage, application and normalization conditions.…”

“…Interaction between antigen and antibody results in the formation of immune complexes, which further interact with other serum components, such as complement, and cellular receptors. In order to generate a realistic picture about the events ensuing antigen recognition by antibody, we developed a protein microarray-based technique (Papp et al, 2007). On-chip complement activation generates functional antibody profiles and helps characterize immunity in a systematic way (Papp et al, 2008a;Papp et al, 2008b).…”