A human monoclonal antibody against the collagen type IV α3NC1 domain is a non- invasive optical biomarker for glomerular diseases  Kapil Chaudhary, Daniel T. Kleven, Tracy L. McGaha, Michael P. Madaio  Kidney International  Volume 84, Issue 2, Pages 403-408 (August 2013) DOI: 10.1038/ki.2013.99 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Human monoclonal antibody F1.1 binds to exposed anti-glomerular basement membrane (GBM) epitopes. (a) Unfixed healthy mouse (A–E) and human (F–J) kidney sections (4μm) were treated with acid-urea (B, G), Gu-HCL (C, H), chymotrypsin (D, I), and H2O2 (E, J) and incubated with fluorophore conjugated F1.1. Chemical or proteolytic treatment facilitated binding of F1.1 to the GBM, which was not detected on untreated sections of either mouse or human kidneys (A, F). (b) Unfixed frozen kidney sections from patients (see Supplementary Table S1 online) were probed with fluorophore conjugated F1.1 by direct immunofluorescence. (a) mouse kidney= × 100, human kidney= × 60; (b) magnifications= × 25. Experiments were repeated multiple times with similar results. Kidney International 2013 84, 403-408DOI: (10.1038/ki.2013.99) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Non-invasive optical in vivo quantification of exposed anti-glomerular basement membrane (GBM) epitopes by the human monoclonal antibody F1.1. (a) Nephrotoxic serum nephritis (NTN), puromycin aminoglycoside nephrosis (PAN), and control (phosphate-buffered saline (PBS)) mice were injected with either infrared, fluorophore tagged F1.1 or isotype immunoglobulin G (IgG) control, and live animal imaging was performed. (b) Kidneys from the same experiment were removed and imaged as described in Materials and Methods. Only NTN and PAN mice showed kidney-specific fluorescence. (c) Frozen kidney sections (4μm) from same experiment; F1.1 detection by goat anti-human antibody (red). Note—GBM-specific staining of F1.1 in NTN and PAN mice. (d) Intensity of kidney-specific fluorescence was quantified and plotted (bar diagram for each group). Body weight (e), blood urea nitrogen (f), and urine protein/creatinine ratios (g) were quantified in mice injected with 15μg/g of either F1.1 or isotype control human IgG (IgG2 kappa). (h) Representative histology of kidney section from mice injected with 15μg/gm of HumAb F1.1. (i) or 15μg/gm of isotype control human IgG (IgG2 kappa, ii) showing no evidence of nephritis. For all graphs values represent mean value±s.d. **P>0.01 as determined by Student’s t-test. All experiments were repeated at least twice with similar results. N=5 mice per group. d, day. Kidney International 2013 84, 403-408DOI: (10.1038/ki.2013.99) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 F1.1 signal in vivo correlates with the severity of nephritis, and disease progression or resolution. Swiss mice were injected with high-dose (20μg/g; a, b) or low-dose (3μg/g; c, d) nephrotoxic sheep immunoglobulin G (IgG) intraperitoneally or normal sheep IgG and the glomeruli were imaged with F1.1 in vivo. (b, d) At the times indicated, urine protein/creatinine ratios and blood urea nitrogen (BUN) levels were determined. Data points represent mean values for groups±s.d. *P>0.05 and **P>0.01 as determined by Student’s t-test. All experiments were repeated three times with similar results. N=5 mice per group. d, day; NTS, nephrotoxic serum. Kidney International 2013 84, 403-408DOI: (10.1038/ki.2013.99) Copyright © 2013 International Society of Nephrology Terms and Conditions