The BDL experiments were approved by the Experimental Animal Committee of the Danish Ministry of Justice and were performed according to the European Standard for Good Clinical Practice (2008/561-1450).
The CCl4 study was approved by the Ethical Committee of Animal Experimentation of the University of Barcelona (B-NNP-233/09) and was performed according to the criteria of the Investigation and Ethics Committee of the Hospital Clinic Universitari (Barcelona, Spain).
All reagents used for the experiments were standard high-quality chemicals from companies such as Merck (Whitehouse Station, NJ, USA) and Sigma Aldrich (St.Louis, MO, USA). The synthetic peptides used for monoclonal antibody production were purchased from the Chinese Peptide Company, Beijing, China.
In vitro cleavage
Purified type VI collagen from human placenta (cat. no. ab7538, Abcam, Cambridge, UK) was cleaved with pro-MMP-2 or pro-MMP-9 (cat. no. 444213; 444231; Calbiochem, Merck, Whitehouse Station, NJ, USA). Fifty µg MMP-2 or MMP-9 was activated with 20 µl 1 mM 4-aminophenylmercuric acetate (AMPA) in dimethyl sulfoxide and incubated at 37°C for 3 hours. Type VI collagen was delivered dissolved in 0.5 M acetic acid. To facilitate MMP cleavage, the protein was dialyzed for two days to remove the acetic acid. The liquid was filtered to remove proteins below 10 kDa (Microcon Ultracel YM-10, cat. no. 42407, Millipore, Billerica, MA, USA). Each MMP cleavage was performed separately by mixing 100 µg type VI collagen and 1 µg of either MMP-2 or MMP-9 in MMP buffer (100 mM Tris-HCl, 100 mM NaCl, 10 mM CaCl2, 2 mM Zn acetate, pH 8.0). As control, 100 µg of collagen was mixed with MMP buffer alone. The solutions were incubated for 24 hours at 37°C. The cleavage reaction was stopped using 50 µM ethylenediaminetetraacetic acid (EDTA) to a final concentration of 1 µM. Cleavage was verified by visualization using the SilverXpress® Silver Staining Kit (cat. no. LC6100, Invitrogen, Carlsbad, Ca, USA) according to the manufacturer's instructions.
Pepsin cleavage was performed by mixing 100 µg type IV collagen and 1 µg of pepsin in pepsin buffer (0.2 M sodium acetate buffer, pH 4.0). The resultant enzyme/protein mixture was incubated at 37°C for 2 hours. At the designated time, 2 M Trizma Base was added to adjust pH to neutral to stop the reactions.
Peptide fragments in the in vitro cleaved samples were identified using liquid chromatography (LC) coupled to electrospray ionization (ESI) tandem mass spectrometry (LC-MS/MS). LC-MS samples were ultra-filtrated to remove proteins above 10 kDa, the pH was adjusted to 2.0 using formic acid, and a 4 µl sample was analyzed by LC-MS/MS. LC was performed on a nanoACQUITY UPLC BEH C18 column (Waters, Milford, MA, USA) using a formic acid/acetonitril gradient. MS and MS/MS were performed on a Synapt High Definition Mass Spectrometry quadruple time of flight MS (QUAD-TOF; Waters, Milford, MA, USA), with an acquisition range of 350–1600 m/z in MS and 50–2000 m/z, in MS/MS. The software “ProteinLynx Global SERVER (PLGS)” (Waters, Milford, MA, USA) was used to analyze spectra and generate peak lists. To identify peptides, MS and MS/MS data was searched against a type VI collagen (FASTA) protein database using the Mascot 2.2 (Matrix Science, Boston, MA, USA) software with the ESI-QUAD-TOF settings and carbamidomethyl (C), oxidation of methionine (M), oxidation of lysine (K) and oxidation of proline (P) as variable modifications.
The six amino acids in the N- or C-terminal of the peptides identified by MS were regarded as a neo-epitope generated by the protease in question. All protease-generated sequences were analyzed for homology and distance to other cleavage sites and tested for homology using NPS@: network protein sequence analysis (Combet C, Blanchet C, Geourjon C, Deleage G. NPS@:network protein sequence analysis. Trends Biochem Sci 2000; 25: 147–50).
The peptide conjugation was performed using the Maleidide Activated Immunogen Conjugation Kit (Sigma-Aldrich, MO, USA). Briefly, the cysteine-containing immunogenic neo-epitope (YRGPEGPQGP-GGC, 400 µl peptide at 5 mg/ml) with one free sulfhydryl (-SH) group was mixed in conjugation buffer with the maleimide-activated ovalbumin (OVA) (180 µl OVA at 10 mg/ml) as a carrier protein with an available maleimide group that could react with sulfhydryl-containing peptides and incubated for 2 hours at room temperature. Conjugated products were cleared of EDTA and sodium azide by desalting or dialysis for two days. For the biotin-conjugated peptides, the biotin-conjugated lysine was added in the solid-phase peptide synthesis procedure.
Monoclonal antibody development
4–6 weeks-old Balb/C mice were immunized subcutaneously with about 200 µl emulsified antigen and 50 µg of the neo-epitope CO6-MMP (YRGPEGPQGP-GGC-OVA). Consecutive immunizations were performed at 2-week intervals until stable sera titer levels were reached in Freund's incomplete adjuvant. Blood samples were collected from the 2nd immunization. At each blood sampling, the serum titer was determined and the mouse with highest anti-serum titer was selected for fusion. After the 4th immunization, this mouse was rested for 1 month and then boosted intravenously with 50 µg CO6-MMP in 100 µl 0.9% sodium chloride solution three days before isolation of the spleen for cell fusion.
Fusion and antibody screening
The fusion procedure performed as described by Gefter et al 
. Briefly, mouse spleen cells were fused with SP2/0 myeloma fusion partner cells. The hybridoma cells were cloned using a semi-solid medium method and transferred into 96-well microtiter plates for further growth and incubated in a CO2
-incubater. Standard limited dilution was used to promote monoclonal growth. Supernatants were screened using an indirect ELISA with streptavidin-coated microtitre plates and YRGPEGPQGP-K-Biotin as a capture peptide.
Characterization of clones
Native reactivity and peptide binding of the monoclonal antibodies was evaluated by displacement of native samples (human/rat/mouse serum, plasma and urine) in a preliminary ELISA using 10 ng/mL biotinylated peptide coater on a streptavidin-coated microtitre plate and the supernatant from the growing monoclonal hybridoma. Specificities of the clones to a free peptide (YRGPEGPQGP), a non-sense peptide, and an elongated peptide (GYRGPEGPQG) were tested. Isotyping of the monoclonal antibodies was performed using the Clonotyping System-HRP kit, cat. no. 5300-05 (Southern Biotech, Birmingham, AL, USA). The selected clones were purified using protein G columns according to manufacturer's instructions (GE Healthcare Life Science, Little Chalfont, Buckinghamshire, UK). Selected monoclonal antibodies were labeled with horseradish peroxidase (HRP) using the Lightning link HRP labeling kit according to the instructions of the manufacturer (Innovabioscience, Babraham, Cambridge, UK).
CO6-MMP ELISA methodology
In preliminary experiments, we optimized the reagents, their concentrations and the incubation periods by performing several checkerboard analyses. The CO6-MMP ELISA was developed as follows: A 96-well streptavidin plate was coated with biotinylated synthetic peptide YRGPEGPQGP-K-Biotin dissolved in assay buffer (25 mM Tris, 1% BSA, 0.1% Tween-20, pH 7.4) and incubated 30 minutes at 20°C. Twenty µl of peptide calibrator or sample were added to appropriate wells, followed by 100 µL of conjugated monoclonal antibody and incubated 1 hour at 20°C. Finally, 100 µL tetramethylbenzinidine (TMB) (Kem-En-Tec cat. no. 438OH) was added and the plate was incubated 15 minutes at 20°C in the dark. All the above incubation steps included shaking at 300 rpm. After each incubation step the plate was washed five times in washing buffer (20 mM Tris, 50 mM NaCl, pH 7.2). The TMB reaction was stopped by adding 100 µl stopping solution (1% HCL) and measured spectrophotometrically at 450 nm with 650 nm as the reference. A standard curve was performed by serial dilution of the CO6-MMP peptide and plotted using a 4-parametric mathematical fit model. Standard concentrations were 0, 0.39, 7.8, 15.6, 31.3, 62.5, 125 250 ng/mL.
From 2-fold dilutions of pooled serum and plasma samples, linearity was calculated as a percentage of recovery of the 100% sample. The lower detection limit (LDL) was calculated from 21 determinations of the lowest standard (the zero standard) and calculated as the mean +3x standard deviation. The inter- and intra-assay variation was determined by 10 independent runs of 5 QC samples, with each run consisting of two replicas of double determinations of the samples. Finally, for each assay, a master calibrator prepared from synthetic peptides accurately quantified by amino acid analysis was used for calibration purposes.
The analyte stability was determined for six serum samples (three rat and three human) for 10 freeze and thaw cycles.
The developed CO6-MMP ELISA was evaluated using 20 µl of the cleavage-samples: type VI collagen, type VI collagen cleaved with MMP-2, type VI collagen cleaved with MMP-9 described under “In vitro cleavage”. The negative control was in vitro cleaved type VI collagen with fibroblast activation protein (FAP). Cross-reactivity was tested using intact or in vitro cleaved type I or IV collagen using 20 µL peptide solution of 1000 ng/mL for each test in the assay. Neo-epitope specificity was tested using cleaved (by either MMP-2 or MMP-9) and non-cleaved type VI collagen and by an elongated CO6-MMP amino acid sequence (GYRGPEGPQG).
Bile duct ligation
A total of 40 female Sprague-Dawley rats aged 6 months were housed at the animal research facilities at Nordic Bioscience, Denmark. The rats were kept in standard type III-H cages at 18–22°C with bedding and nest material (Altromin 1324; Altromin, Lage, Germany) and water ad libitum. Rats were kept under conditions of a 12-hour light: dark cycle. Experiments began after 1 week of acclimatization. Bile duct ligation (BDL) was performed in anaesthetized rats by ligation of the bile duct in two places and dissection between the ligations in an open-surgery procedure. In sham-operated rats, the abdomen was closed without BDL. BDL- or sham-operated rats were sacrificed after 2 or 4 weeks.
The study included 52 3-months old male Wistar rats treated with CCl4
and 28 Wistar control rats (Charles-River, Saint Aubin les Elseuf, France). Complete details of the study are described elsewhere (Segovia-Silvestre T et al.). Liver damage was induced as previously described 
, and in short included administration by inhalation of CCl4
twice weekly. Phenobarbital (0.3 g/l) was added to the drinking water. Animals were stratified into groups receiving 8, 12, 16 or 20 weeks of CCl4
13 for CCl4
7 control for each group). Control rats received Phenobarbital only. Four animals from the CCl4
groups died during the study. After the stated weeks of CCl4 administration the rats were weighed, anesthetized with pentobarbital (50 mg/kg) and terminated by decapitation.
Blood and tissue sampling
Blood samples were taken under light CO2/O2 anesthesia at baseline and at termination from the retro-orbital sinus of rats which had fasted for at least 14 hours. The collected blood was left for 30 min at room temperature to clot, followed by centrifugation at 3000 g for 10 min. All clot-free liquid was transferred to new tubes and centrifuged again at 3000 g for 10 min. The serum was then transferred to clean tubes and stored at −80°C.
Livers were carefully dissected, weighed, fixed in 4% formaldehyde for a minimum of 24 hours, cut into appropriate slices and embedded in paraffin. Liver sections (4–5 µm thick) were stained with 0.1% Sirius Red F3B (Sigma-Aldrich, St. Louis, MO) in saturated picric acid (Sigma-Aldrich).
Histology image analysis
Relative fibrosis area (expressed as a percentage of total liver area) was assessed by analyzing 36 fields of Sirius Red-stained liver sections per animal. Each field was acquired at 10× magnification [E600 microscope (Nikon) and RT-Slider SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, Michigan, US)]. Results were analyzed using a computerized Bioquant Life Science morphometry system. To evaluate the relative fibrosis area, the measured collagen area was divided by the net field area and then multiplied by 100. Subtraction of vascular luminal area from the total field area yielded the final calculation of the net fibrosis area. From each animal analyzed, the amount of fibrosis as a percentage was measured and the average value presented 
Liver sections (4 µm) were de-paraffinised, hydrated and further peroxidase activity was blocked with the addition of 0.4% hydrogen peroxide. Sections were then incubated with a polyclonal antibody against type VI collagen (1
100; Abcam, Cambridge, UK). Sections were then rinsed and the antibody binding was depicted using the Super Sensitive Polymer-HRP IHC Detection System combined with AEC substrate, according to the supplier's instructions (Biogenex, Taby, Sweden). Sections were counterstained with Mayer's haematoxylin. Digital photographs were taken using an Olympus B×60 microscope with ×40 magnification and an Olympus 5050-zoom digital camera (Olympus, Tokyo, Japan).