All chemicals, enzymes and cell culture reagents were purchased from Sigma-Aldrich (Copenhagen, Denmark), or VWR (Herlev, Denmark), if not otherwise stated. The ELISA plates, pre-coated with streptavidin (Nunc clear 96-well plates), were purchased from Roche Diagnostics (Mannheim, Germany). Immunogens, standard and coating peptides were purchased from the Chinese Peptide Company (Beijing, China) and from American Peptide (Sunny Valley, CA, USA).
In vitro peptide generation
The BGM neo-epitope was identified by in vitro degradation of bovine articular cartilage purified biglycan (Sigma-Aldrich, Copenhagen, Denmark) by MMP-9 and -12. The purified biglycan had been filtered to remove proteins below 10,000 kDa (Microcon Ultracel YM-10, catalog number. 42407, Millipore, Billerica, MA, USA) and had not been de-glycosylated prior to MMP digestion. The buffer used for the MMP cleavage of biglycan (1 mg/mL) consisted of 100 mM Tris–HCl, 100 mM NaCl, 10 mM CaCl2 and 2 mM ZnAc, at pH 8.0. The cleavage fragments were obtained after 72 hours of incubation with each protease. As a control biglycan (1 mg/mL) was incubated with MMP-buffer. The cleavages were stopped by 5 mM EDTA and verified by SDS-PAGE.
Peptide identification and antibody generation
After the in vitro cleavage, peptides of biglycan were identified using liquid chromatography coupled to electrospray ionization (ESI) tandem mass spectrometry (LC-MS/MS) as previously described . To identify peptides, MS and MS/MS data were searched against a biglycan (FASTA) protein database using the Mascot 2.2 (Matrix Science, Boston, MA, USA) software with ESI-QUAD-TOF settings and carbamidomethyl (C), oxidation of methionine (M), oxidation of lysine (K) and oxidation of proline (P) as variable modifications. The first six amino acids of each free end of the protease-generated peptide sequences identified by MS were regarded as a neo-epitope generated by the specific protease.
All MMP-9 and -12 generated neo-epitopes were analyzed for distance to other cleavage sites and then blasted for protein and species homology using the NPS@: network protein sequence analysis . Among all the different neo-epitopes, the sequence 344YWEVQPATFR353 was chosen according to the mentioned criteria. A monoclonal antibody targeted against the N-terminal part of the selected peptide was generated as previously described .
BGM ELISA development
A competitive ELISA for the biglycan selected neo-epitope BGM was developed as follows: a 96-well streptavidin-coated plate was coated with 2.5 ng/mL biotinylated synthetic peptide YWEVQPATFR-K-Biotin dissolved in PBS buffer (2 mM KH2PO4, 9 mM Na2HPO4, 2H2O, 3 mM KCl, 137 mM NaCl, pH 7.4) and incubated for 30 min at 20°C by constant shaking at 300 rpm. 20 μL of peptide calibrator prepared by two-fold pre-dilution of the standard peptide (YWEVQPATFR) starting from 250 ng/mL or sample dissolved in assay buffer (2 mM KH2PO4, 9 mM Na2HPO4, 2H2O, 3 mM KCl, 34 mM NaCl, 5% Osteocalcin EIA Puf-Liq (Roche Diagnostics Deutschland GmbH) pH 7.4) were added to appropriate wells, followed by 100 μL of 40 ng/mL peroxidase-labeled NB202-7 9D6 antibody and incubated for one hour at 20°C by constant shaking at 300 rpm. Finally, 100 μL of tetramethylbenzidine (TMB) (catalog number 438OH; Kem-En-Tec, Copenhagen, Denmark) were added, and the plate was incubated for 15 minutes at 20°C in the dark and shaken 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 of stopping solution (1% HCl) and the colorimetric reaction was measured at 450 nm with reference at 650 nm on a standard laboratory plate reader. Data were acquired with the SoftMax Pro v5.0 (Molecular devices, LLC, Sunnyvale, CA, USA) program.
Technical evaluation of BGM assay
Technical assay validation was performed according to international guidelines of assay development. Briefly, linearity was calculated as a low, medium or high percentage of recovery of the 100% sample from two-fold dilutions of quality control (QC) human serum and from rat serum. The lower limit of detection (LDL) was determined from 21 zero samples (buffer only) and calculated as the mean + 3 x standard deviation. Percentage dilution recovery was calculated as the mean of five human serum and five human plasma samples, four rat serum and four rat plasma and three mouse serum and three mouse plasma diluted 1:2 and 1:4. Inter- and intra-assay variations were calculated as the mean variation between ten individual determinations of eight QC samples (human serum) with each run consisting of two replicas of double determinations of the samples.
The developed BGM ELISA was evaluated using 20 μL of the samples: intact biglycan, biglycan cleaved with MMP-9, biglycan cleaved with MMP-12, the standard BGM peptide YWEVQPATFR and the BGM peptide elongated at the N-terminal end with one amino acid (PYWEVQPATFR). Specificity was tested using a non-sense peptide NNQIDHIDEK and a non-sense coater Biotin-K- NNQIDHIDEK.
Bovine cartilage explant cultures
Bovine cartilage explants (BEX) were harvested by dissecting the outermost layer of articular cartilage from bovine knee joints, as previously described . The cartilage explants (16 ± 4 mg) were placed in 96-well plates and incubated at 37°C, with 5% CO2 and shaken at 50 rpm under serum-free conditions (n = 5 per condition). Each explant was cultured in 200 μl of DMEM for seventeen days, with the medium being changed every three to four days, under one of the following conditions: 1) Without catabolic factors (W/O), 2) Metabolically inactivated by liquid nitrogen, 3) With the catabolic cytokines oncostatin M (10 ng/mL) and TNF-α (20 ng/mL), O+T to stimulate MMP activity; 4) O+T supplemented by the MMP inhibitor GM6001 (10 μM); and 5) O+T supplemented by the cysteine protease inhibitor E64 (50 μM), here used as a negative control, as the selective cathepsin inhibitor should not have an effect on MMP activity. Each condition was replicated five times. The metabolic activity (viability) of the articular explants was quantitatively measured on the last day in culture, using the Alamar Blue assay (TREK Diagnostic Systems, LTD. East Grinstead, West Sussex RH19 1XZ UK) according to the manufacturer’s instructions (data not shown).
Collagen-induced arthritis (CIA) model
Levels of BGM were measured in a CIA rat model. Complete details of the study have been previously described . The animal experiment protocol was approved by the local animal ethics committee at Nordic Bioscience Beijing. The ethical approval number is NBB-AM-R/2009-01. Briefly, CIA was induced in ten seven-week old female Lewis rats by immunizing with 450 μl 2 mg/mL porcine type II collagen dissolved in 0.05 M acetic acid and emulsified 1:1 in incomplete Freund’s adjuvant on day 0 and 7. Ten Lewis rats, injected only with 0.05 M acetic acid, were used as control. Every day, starting from day 8, rats were examined for visual signs of disease, defined as macroscopic evidence of increase in paw size. The rats were sacrificed on day 26. Serum samples were collected throughout the experiment from overnight fasted animals.
Rat model of CCL4-induced liver fibrosis
Serum BGM levels were measured in a CCL4 inhalation rat model of liver fibrosis. Complete details of the study have been previously described . 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). The study included 52 male Wistar rats treated with CCL4 and 28 male Wistar control rats (Charles-River, Saint Aubin les Elseuf, France). Induction of liver fibrosis was performed as previously described . Briefly, CCL4 was administered by inhalation twice weekly and phenobarbital (0.3 g/l) added to the drinking water. Control rats received phenobarbital only. Animals were stratified into groups receiving CCL4 or control treatment for 8, 12, 16 or 20 weeks (n = 13 for CCL4; n = 7 control for each group). Four animals from the CCL4 groups died during the study. Blood was collected at termination and allowed to stand at room temperature for 30 minutes to allow clotting, before centrifugation at 3,000 g for 10 minutes. All clot-free liquid was transferred to new tubes and centrifuged again at 3,000 g for 10 minutes. Samples were stored at -80°C prior to biomarker assessment. Liver sections (4 μm thick) were stained in 0.1% Sirius red F3B (Sigma-Aldrich, St. Louis, MO, USA) in saturated picric acid (Sigma-Aldrich St. Louis, MO, USA). From each animal analyzed, the amount of fibrosis was expressed as a percentage of the total liver area of 36 fields and the average value is presented. Each field was acquired at 10 x magnification .
Rat model of bile duct ligation (BDL) induced liver fibrosis
Serum BGM levels were measured in a rat model of liver fibrosis induced by bile duct ligation. Complete details of the study have been previously described [18, 35]. The BDL experiment was approved by the Experimental Animal Committee of the Danish Ministry of Justice and was performed according to the European Standard for Good Clinical Practice (2008/561-1450).
The study included a total of 81 female Sprague–Dawley rats aged six months. Liver fibrosis was induced in anaesthetized rats by standard BDL in which the bile duct was ligated in two places and dissected between the ligations prior to closing the abdomen. In sham-operated rats, the abdomen was closed without BDL. The rats were divided into four groups: group 1 (10 BDL, 8 sham) was sacrificed after one week, group 2 (12 BDL, 8 sham) sacrificed after two weeks, group 3 (13 BDL, 8 sham) sacrificed after three weeks, and group 4 (14 BDL, 8 sham) sacrificed after four weeks. During the four weeks, 15 of 81 rats, 14 of them BDL operated, were terminated due to excessive weight loss.
The ELISA standard curve was fitted by the four-parameter method:
Comparison between measurements of biomarkers in culture supernatants and differences between tertiles were assessed by one-way ANOVA with Dunnett’s post-test assuming Gaussian distribution, on accumulated data. Comparison of two subject groups was made using the non-parametric Mann–Whitney test, α = 0.05. The correlations coefficient was calculated using the Spearman’s ρ non-parametric test. GraphPad Prism v.5 (GraphPad Software, Inc. La Jolla, CA 92037 USA) was used for drawing graphs and calculating statistics.