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

Abstract

Humoral immune responses are traditionally characterized by determining the presence and quality of Abs specific for certain Ags. Arraying of large numbers of Ags allows the parallel measurement of Abs, generating patterns called Ab profiles. Functional characterization of these Abs could help draw an even more informative map of an immune response. To generate functional Ab profiles we simultaneously tested not only IgM, IgG, and IgA binding to, but also complement activation by, a panel of endogenous and exogenous Ags printed as microarrays, using normal and autoimmune human sera. We show that complement activation by a particular Ag in a given individual cannot be predicted by the measurement of Ag-specific Abs, despite a general correlation between the amount of Ag-bound Ab and the deposited C3 fragments. This is due to both differences in the isotypes that dominate in the recognition of an Ag and individual variations for a given isotype, resulting in altered complement activation potential. Thus, Ag-specific C3 deposition can be used as an additional parameter in immune response monitoring. This is exemplified by comparing the coordinates of Ags, used for the diagnosis of systemic lupus erythematosus, of normal and autoimmune serum samples in a two-dimensional space derived from C3 deposition and Ab binding. Since cleavage fragments of C3 mediate important immunological processes, we propose that measurement of their deposition on Ag microarrays, in addition to Ab profiling, can provide useful functional signature about the tested serum.

Complementing antibody profiles: Assessing antibody function on antigen microarrays

“…Experimental data, in agreement with the expectable correlations [15] suggested that it is mostly IgM and IgG that determines complement depositions to self antigens. Detection of these two classes of immunoglobulins seems inevitable in order to interpret effector functions.…”

Preliminary comparison of different immune and production components in local and imported Saanen goats reared under a sub-tropical environment

“…The CF titer increased significantly at 4 weeks following priming with c-RBC in local compared to that of the Saanen does (p<0.05); however, the difference in CF titers was insignificant at 3 weeks following boosting (p>0.05). The reasons for the difference in the CF titer following priming in locals versus Saanen does will be the subject of future investigations due to the importance of CF in amplification of the humoral responses (Paul 2008), in chemotaxis (Meyers 2007) and anaphylatoxic shocks (McEwen 1992;Decker 2000;Papp et al 2008), and in opsonization (Rooijakkers et al 2005;Paul 2008). …”

Functional autoantibodies against SSX-2 and NY-ESO-1 in multiple myeloma patients after allogeneic stem cell transplantation

“…First, we asked whether our patients' anti-NY-ESO-1 or anti-SSX-2 antibodies, which reacted not only with the respective recombinant proteins but also recognized antigen endogenously expressed by tumor cell lines (Supplementary Figure 3C) were in principle able to directly activate the complement system. Assessing the activation of the classical complement pathway by serum antibodies [10], we found for the first time, both CTA-specific antibodies to be potent inducers of the complement system, as evidenced by C3-deposition on a membrane coated with recombinant protein (Fig. 2a).…”

“…Proteins were blotted and membrane treated as outlined above. As described by Papp et al [10], this deposit can be detected on nitrocellulose membranes by a C3-specific antibody. Human serum was diluted 1:5 and incubated for 3 h on the nitrocellulose membrane at room temperature prior to stringent washing.…”