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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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 8

Clones 3d8b, 3d9a, and 3d29 bind to mouse C3 fragments generated in vivo.

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Clones 3d8b, 3d9a, and 3d29 bind to mouse C3 fragments generated in vivo...
(A) Kidney tissue sections from factor H–deficient mice (fH–/–) were used to test binding of the antibodies to C3 tissue deposits. Factor H mice are known to have abundant deposition of C3 fragments along the glomerular capillaries without IgG at this location. This was confirmed by immunostaining with a polyclonal antibody against mouse C3. Kidney tissue sections were then incubated with 5 μg/ml of each clone. Clones 3d8b, 3d9, and 3d29 bound to the capillaries in a pattern identical to that of polyclonal anti-C3. The remaining 6 clones did not demonstrate substantive binding (the result for clone 3d31 is shown). (B) We immunostained kidneys from factor I–deficient (fI–/–) mice with a polyclonal antibody against C3 and with mAb 3d29. The fI–/– mice cannot generate iC3b. The absence of glomerular staining in fI–/– mice by mAb 3d29 confirms that the mAb does not recognize C3b. Glomeruli are indicated with arrowheads. Original magnification, ×400 for all panels, including the inset.