Reagents

All reagents used for the experiments were standard high-quality chemicals obtained 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 IV collagen from human placenta (cat. no. 11-511-248457; GenWay Biotech, Inc., San Diego, CA, USA) was cleaved with pro-MMP-2 (cat. no. 444213; Calbiochem, Gibbstown, NJ, USA) or pro-MMP-9 (cat. no. 444231; Merck, Whitehouse Station, NJ, USA). Fifty micrograms of MMP-2 or MMP-9 were activated with 20 μL of 1 mM 4-aminophenylmercuric acetate in dimethyl sulfoxide and incubated at 37°C for three hours. Type IV 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 (cat. no. 42407, Microcon Ultracel YM-10; Millipore, Billerica, MA, USA). Each MMP cleavage was performed separately by mixing 100 μg of type IV collagen and 10 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 a control, 100 μg of collagen was mixed with MMP buffer alone. The solutions were incubated for two 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.

Peptide identification

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 ultrafiltrated 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/acetonitrile gradient. MS and MS/MS were performed on a Synapt G1 High Definition Mass Spectrometer quadrupole time-of-flight MS (QUAD-TOF; Waters) with an acquisition range of 350 to 1,600 m/z in MS and 50 to 2000 m/z in MS/MS. ProteinLynx Global SERVER software (Waters) was used to analyze spectra and generate peak lists. To identify peptides, MS and MS/MS data were searched against a type IV collagen protein database (FASTA) using Mascot 2.2 software (Matrix Science, Boston, MA, USA) 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 neoepitope 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 [13].

Peptide conjugation

The peptide conjugation was performed using the Maleimide Activated BSA, KLH Conjugation Kit (Sigma-Aldrich). Briefly, the cysteine-containing immunogenic neoepitope (CGG-GTPSVDHGFL, 400 μL of peptide at 5 mg/mL) with one free sulfhydryl group (-SH) was mixed in conjugation buffer containing the maleimide-activated ovalbumin (OVA) (180 μL of OVA at 10 mg/mL) as a carrier protein with an available maleimide group that could react with -SH-containing peptides and incubated for two hours at room temperature. Conjugated products were cleared of EDTA and sodium azide by desalting or dialysis for two days. The biotin-conjugated lysine was added to the biotin-conjugated peptides in the solid-phase peptide synthesis procedure.

Monoclonal antibody development

Four- to six-week-old Balb/c mice were immunized subcutaneously with about 200 μL of emulsified antigen and 50 μg of the neoepitope CO4-MMP (OVA-CGG-GTPSVDHGFL). Consecutive immunizations were performed at two-week intervals until stable sera titer levels were reached in Freund's incomplete adjuvant. Blood samples were collected from the second immunization. For each blood sampling, the serum titer was determined and the mouse with the highest antiserum titer was selected for fusion. After the fourth immunization, this mouse was rested for one month and then boosted intravenously with 50 μg of CO4-MMP in 100 μL of 0.9% sodium chloride solution three days before isolation of the spleen for cell fusion.

Fusion and antibody screening

The fusion procedure was performed as described by Gefter et al. [14]. Briefly, mouse spleen cells were fused with SP2/0 myeloma fusion partner cells. The hybridoma cells were cloned using a semisolid medium method, transferred into 96-well microtiter plates for further growth and incubated in a CO₂ incubator. Standard limited dilution was used to promote monoclonal growth. Supernatants were screened using an indirect ELISA with streptavidin-coated microtiter plates and biotin-CGG-GTPSVDHGFL as a capture peptide.

Characterization of clones

Native reactivity and peptide binding of the monoclonal antibodies were 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 microtiter plate and the supernatant from the growing monoclonal hybridoma. Specificities of the clones to a free peptide (GTPSVDHGFL), a non-sense peptide and an elongated peptide (TPSVDHGFLV) 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 the manufacturer's instructions (GE Healthcare Life Sciences, Little Chalfont, UK). Selected monoclonal antibodies were labeled with horseradish peroxidase (HRP) using the Lightning-Link Horseradish Peroxidase labeling kit (Innova Biosciences, Cambridge, UK) according to the instructions of the manufacturer.

CO4-matrix metalloproteinase enzyme-linked immunosorbent assay methodology

In preliminary experiments, we optimized the reagents, their concentrations and the incubation periods by performing several checkerboard analyses. The CO4-MMP ELISA was developed as follows: A 96-well streptavidin plate was coated with biotinylated synthetic peptide biotin-CGG-GTPSVDHGFL, dissolved in PBS buffer (2 mM KH₂PO₄, 9 mM Na₂HPO₄, 2H₂O, 3 mM KCl, 137 mM NaCl, pH 7.4) and incubated for 30 minutes at 20°C by constant shaking at 200 rpm. Twenty microliters of peptide calibrator or sample dissolved in assay buffer (25 mM Tris, 1% BSA, 0.1% Tween 20, pH 7.4) were added to appropriate wells, followed by 100 μL of conjugated monoclonal antibody, and incubated for one hour at 20°C by constant shaking at 300 rpm. Finally, 100 μL of tetramethylbenzidine (TMB) (cat. no. 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 measured spectrophotometrically at 450 nm with 650 nm as the reference. A standard curve was performed by serial dilution of the CO4-MMP peptide and plotted using a four-parametric mathematical fit model. Standard concentrations were 0, 1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 ng/mL.

Technical evaluation

From twofold 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 3 × standard deviation. The inter- and intraassay variations were determined by ten independent runs of five QC samples, with each run consisting of two replicates of double-determinations of the samples. Spiking recovery was determined by comparing different concentrations of a peptide sample in buffer and in human serum. The peptide sample was the type IV collagen cleaved with MMP-9 and pepsin. 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 ten freeze-thaw cycles.

Enzyme-linked immunosorbent assay characterization

The developed CO4-MMP ELISA was evaluated using 20 μL of the samples: intact type IV collagen, type IV collagen cleaved with pepsin, type IV collagen cleaved with MMP-2, type IV collagen cleaved with MMP-9, type IV collagen cleaved with MMP-9 and pepsin, and an elongated CO4-MMP amino acid sequence (TPSVDGHFLV). Cross-reactivity was tested using in vitro cleaved collagen type I or type VI.

Bile duct ligation

A total of 81 female Sprague-Dawley rats, age six months, were housed at the animal research facilities at Nordic Bioscience A/S, Herlev, Denmark. The 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 rats were housed in standard cages at 18°C to 22°C with bedding and nest material (1324 TPF; Altromin Spezialfutter GmbH & Co. KG, Lage, Germany) and tap water ad libitum. The rats were kept under 12-hour light-dark cycle conditions. Experiments began after one week of acclimatization. Liver fibrosis was induced in anesthetized 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: rats in group 1 (ten BDL and eight sham) were killed after one week, rats in group 2 (12 BDL and 8 sham) were killed after two weeks, rats in group 3 (13 BDL and 8 sham) were killed after three weeks and rats in group 4 (14 BDL and 8 sham) were killed after four weeks.

Carbon tetrachloride inhalation

The study included 52 three-month-old male Wistar rats treated with CCl₄ and 28 Wistar control rats (Charles River Laboratories, Saint-Aubin-lès-Elbeuf, France). Complete details of the study are described elsewhere [15]. Liver damage was induced as previously described [16] and, in short, included 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 (for each group, n = 13 CCl₄ and n = 7 control). Control rats received phenobarbital only. After the stated weeks of CCl₄ administration, the rats were weighed, anesthetized with pentobarbital (50 mg/kg) and killed by decapitation. The study was performed according to the criteria of the Investigation and Ethics Committee of the Hospital Clinic Universitari (Barcelona, Spain).

Blood and tissue sampling

Blood samples were taken under light CO₂/O₂ anesthesia at baseline and at death from the retroorbital sinuses of rats that had been fasted for at least 14 hours. The collected blood was left for 30 minutes at room temperature to clot, followed by 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. The serum was then transferred to clean tubes and stored at -80°C.

The 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 to 5 μm thick) were stained with 0.1% Sirius red F3B (Sigma-Aldrich) in saturated picric acid (Sigma-Aldrich).

Histological 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 using an E600 microscope (Nikon Instruments Inc., Melville, NY, USA) equipped with a SPOT RT Slider CCD digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). The results were analyzed using a computerized Bioquant Life Science morphometric software system (Bioquant Image Analysis Corp., Nashville, TN, USA. 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. For each animal analyzed, the amount of fibrosis was measured as a percentage and the average value is presented [17].

Immunohistochemistry

Liver sections (4 to 5 μm) were deparaffinized and 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 IV 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 hematoxylin. Digital photographs were taken using an Olympus B 60 × microscope at ×40 magnification equipped with an Olympus C-5050 Zoom digital camera (Olympus, Tokyo, Japan).

Statistical analysis

Mean values and standard errors of the mean (SEM) were calculated using GraphPad Prism software (GraphPad Software, San Diego, CA, USA), and statistical significance was assessed using the Student's two-tailed paired t-test (α = 0.05) assuming normal distribution or the Mann-Whitney two-tailed nonparametric U test (α = 0.05). The coefficient of correlation (R²) and the corresponding P values were determined by linear regression.

In vitro cleavage and selection of peptides

Fragments of type IV collagen cleaved by MMP-2 or MMP-9 with a statistically significant Mascot score (P < 0.05) were identified. All protease-generated neoepitopes were tested for homology. Among more than 200 MMP-generated neoepitopes, five sequences were selected for immunization, as these sequences are unique to type IV collagen. In addition, the sequences 3 to 5 were found in various animal species. The five sequences selected were (1) 328'PGPPGIVIGT.'337, CO4a1 generated by MMP-9; (2) 333'.IVIGTGPLGE'342, CO4a1 generated by MMP-9; (3) 777'.LQGIRGEPGP'786, CO4a1 generated by MMP-9; (4) 1330'DQGDQGVPGA'1339, CO4a1 generated by MMP-2 or MMP-9; and 1438'GTPSVDHGFL'1447, CO4a1 generated by MMP-2 or MMP-9.

The sequence 1438'GTPSVDHGFL'1447 (CO4-MMP) in the α1 chain of type IV collagen generated by MMP-2 or MMP-9 was selected as having the best technical performance because the antibodies were able to distinguish between cleaved and uncleaved type IV collagen. This sequence was 100% homologous in human, rat and mouse.

Clone characterization

After the fusion between spleen cells and myeloma cells, seven hybridoma cell lines remained. After comparing native reactivity, antibody affinity and stability, the best antibody-producing cell line was chosen. The clone selected was the immunoglobulin G2a subtype. The antibody was reactive to human, rat and mouse plasma and serum (Figure 1).

Technical evaluation

The typical standard curve is presented in Figure 2, which shows a four-parametric fit for the assay. The LDL for the CO4-MMP assay was 0.61 ng/ml. Dilution recovery was within 100% ± 20% (Table 1) for human and rat serum. The mean intra- and interassay variations were 4.8% and 12.1%, respectively (Table 2). Analyte stability was acceptable for 2 × to 10 × freeze-thaw cycles for both rat and human serum (Table 3). The percentages are within 100 ± 20%, except for one determination at 8 × for human serum and one determination at 6 × for rat serum. Finally, spiking recovery of peptide in human serum was acceptable, with recoveries ranging from 103% to 110% (Table 4).

Enzyme-linked immunosorbent assay characterization

To characterize the analytes detected in the assay, different collagens were cleaved with different proteases. Pepsin or MMP-9 alone or together was able to generate the CO4-MMP fragment from collagen type IV alone (Figure 2). In contrast, the fragment was not found in intact type IV collagen or MMP-2 cleaved type IV collagen. Finally, no cross-reactivity was seen between CO4-MMP and MMP-9 cleaved type I or VI collagens, which have high homology with the immunization sequence of type IV collagen. No reactivity was seen against the elongated synthetic peptide, proving neoepitope reactivity (Figure 2).

Evaluations performed in the bile duct ligation study

During the four-week experiment, 15 of 81 rats, 14 of them BDL-operated, were killed because of excessive weight loss. CO4-MMP serum levels increased significantly in all BDL groups compared with baseline (group 1: baseline 35.1 ng/mL, death 64.1 ng/mL, P < 0.05; group 2: baseline 34.2 ng/mL, death 84.9 ng/mL, P < 0.01; group 3: baseline 42.9 ng/mL, death 87.4 ng/mL, P < 0.01; group 4: baseline 40.1 ng/mL, death 88.7 ng/mL, P < 0.001), with the maximum increase from baseline of 248% seen in group 2. CO4-MMP levels did not change significantly in the sham-operated rats (Figure 3).

Evaluations performed in the carbon tetrachloride study

Four rats in the CCl₄ groups died during the study. Among the surviving animals, levels of CO4-MMP were significantly elevated at all time points compared to baseline levels, except at week 8 (week 12: control 24.6 ng/mL, CCl₄ 33.5 ng/mL, P < 0.05; week 16: control 24.5 ng/mL, CCl₄ 36.9 ng/mL, P < 0.01; week 20: control 23.0 ng/mL, CCl₄ 43.3 ng/mL, P < 0.01) (Figure 4). The immunohistochemistry showed that collagen type IV deposition was exclusively in the venous wall and around the space of Disse in control rats (Figure 5a). In contrast, in CCl₄-treated rats, type IV collagen was located along the fibrotic bands (Figures 5b through 5e).