On-chip complement activation adds an extra dimension to antigen microarrays.
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 two-color 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.
Papp
Krisztián
K
Department of Immunology, Eötvös Loránd University, Pázmány Péter s. 1/C, Budapest H-1117, Hungary.
Szekeres
Zsuzsanna
Z
Terényi
Nóra
N
Isaák
Andrea
A
Erdei
Anna
A
Prechl
József
J
D000818:Animals; D000917:Antibody Formation / Q000276:immunology; D000941:Antigens / Q000276:immunology*; D003167:Complement Activation / Q000276:immunology*; D003176:Complement C3 / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D040081:Protein Array Analysis / Q000379:methods*
D000941:Antigens; D003176:Complement C3; D007074:Immunoglobulin G