Detection of complement activation using monoclonal antibodies against C3d
Joshua M. Thurman, … , Bärbel Rohrer, V. Michael Holers
Joshua M. Thurman, … , Bärbel Rohrer, V. Michael Holers
Published April 24, 2013
Citation Information: J Clin Invest. 2013;123(5):2218-2230. https://doi.org/10.1172/JCI65861.
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Technical Advance Immunology

Detection of complement activation using monoclonal antibodies against C3d

Abstract

During complement activation the C3 protein is cleaved, and C3 activation fragments are covalently fixed to tissues. Tissue-bound C3 fragments are a durable biomarker of tissue inflammation, and these fragments have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents. We have generated cross-reactive murine monoclonal antibodies against human and mouse C3d, the final C3 degradation fragment generated during complement activation. We developed 3 monoclonal antibodies (3d8b, 3d9a, and 3d29) that preferentially bind to the iC3b, C3dg, and C3d fragments in solution, but do not bind to intact C3 or C3b. The same 3 clones also bind to tissue-bound C3 activation fragments when injected systemically. Using mouse models of renal and ocular disease, we confirmed that, following systemic injection, the antibodies accumulated at sites of C3 fragment deposition within the glomerulus, the renal tubulointerstitium, and the posterior pole of the eye. To detect antibodies bound within the eye, we used optical imaging and observed accumulation of the antibodies within retinal lesions in a model of choroidal neovascularization (CNV). Our results demonstrate that imaging methods that use these antibodies may provide a sensitive means of detecting and monitoring complement activation–associated tissue inflammation.

Authors

Joshua M. Thurman, Liudmila Kulik, Heather Orth, Maria Wong, Brandon Renner, Siranush A. Sargsyan, Lynne M. Mitchell, Dennis E. Hourcade, Jonathan P. Hannan, James M. Kovacs, Beth Coughlin, Alex S. Woodell, Matthew C. Pickering, Bärbel Rohrer, V. Michael Holers

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Figure 6

Inhibition of the CR2-C3d interaction by anti-C3d mAbs.

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Inhibition of the CR2-C3d interaction by anti-C3d mAbs. (A) A competitio...
(A) A competition ELISA was performed to test whether the anti-C3d mAbs interfere with the binding of a recombinant construct of the 2 N-terminal domains of CR2 (MBP-CR2) and plate-bound C3d. The percentage of MBP-CR2 binding (y-axis) (kept at a constant concentration of 10 μg/ml) to C3d was determined in the presence of individual anti-C3d mAbs (x-axis) at a concentration of 26 μg/ml. Values are normalized to a positive control in which C3d-coated wells were incubated with MBP-CR2 in the absence of anti-C3d mAbs (not shown). Also shown for each sample is a negative control in which the wells were coated with BSA instead of C3d. (B–D) Capacity of the Group 1 mAbs (3d8b, 3d9a, and 3d29) to block MBP-CR2 binding to plate-bound C3d at mAb concentrations ranging from 1.625 to 26 μg/ml. (E) 3d10 did not block the binding of CR2 to plate-bound C3d over the same concentration range.