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 2

Generation of mAbs that recognize C3 activation fragments.

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Generation of mAbs that recognize C3 activation fragments. Anti-human C3...
Anti-human C3d hybridomas were generated. (A) The hybridomas were screened against recombinant human C3d by ELISA, and 9 of the clones bound to the protein (clone 7C10 was used as a positive control, and the remaining clones were newly identified). (B) Reactivity of the clones against reduced intact human C3 and recombinant human C3d by Western blot analysis was tested. Three patterns of reactivity were seen: Group 1 clones bound strongly to reduced C3d; Group 2 clones bound to the α chain of reduced intact C3; and Group 3 clones did not bind well to either moiety. The asterisk denotes the mAb whose results are shown. The right-most blot shows the result using a polyclonal antibody against mouse C3. The lower molecular weight bands detected by the mAbs in the C3 samples are likely contaminants. (C) Clone 3d11 recognized all of the human C3 α chain fragments by Western blot analysis. The appearance of the α, α′, α′1, C3dg, and C3d fragments from purified human proteins are shown. The lower molecular weight bands detected in the C3 and iC3b samples are likely contaminants. (D) Immunoprecipitation of C3 fragments in mouse serum demonstrated that the Group 1 clones recognize the iC3b form (α′1 chain) and C3dg, but do not bind to the C3 and C3b (α and α′ chains). Clone 3d16 demonstrated some binding to the iC3b and C3dg fragments. The results using 3d8b were from a separate gel. The immunoprecipitated proteins were visualized by Western blot analysis with mAb 3d11 under reducing conditions.