Collagen type XVI assay
阅读说明：本技术 Xvi型胶原蛋白测定 (Collagen type XVI assay ) 是由 C·詹森 J·H·莫特森 N·维卢姆森 M·A·卡瑞斯戴尔 于 2018-10-19 设计创作，主要内容包括：本发明涉及一种XVI型胶原蛋白测定法及其在评估与XVI型胶原蛋白有关的疾病(特别是结肠直肠癌和溃疡性结肠炎)中、并且用于鉴定患有(或可能发展为)纤维狭窄的5种克罗恩病患者亚组的用途。(The present invention relates to a collagen type XVI assay and its use in the assessment of diseases associated with collagen type XVI, in particular colorectal cancer and ulcerative colitis, and for identifying a subgroup of 5 crohn's disease patients suffering from (or likely to develop) fibrostenosis.)
1. A method of detecting type XVI collagen, or a fragment thereof, in a sample of human biological fluid, the method comprising:
a) obtaining a biological fluid sample from a human patient; and
b) contacting the biological fluid sample with a peptide having the C-terminal amino acid sequence PMKTMKGPFG (SEQ ID NO:1) and detecting binding between the biomarker and the antibody.
2. The method of claim 1, wherein the detecting is quantitative.
3. The method of claim 1, wherein a monoclonal antibody directed against a synthetic peptide having amino acid sequence PMKTMKGPFG (SEQ ID NO:1) is raised.
4. The method of claim 1, wherein the monoclonal antibody does not specifically recognize or bind a C-extended version of the C-terminal amino acid sequence or a C-truncated shortened version of the C-terminal amino acid sequence.
5. The method of claim 1, wherein a measured amount of binding between the monoclonal antibody and the C-terminal biomarker of 1.0ng/mL or greater indicates that the human patient has or is likely to develop ulcerative colitis or colorectal cancer.
6. The method of claim 1, wherein the human patient has medical symptoms or signs indicative of colorectal cancer or ulcerative colitis.
7. The method of claim 1, wherein the human patient is a patient with crohn's disease, and wherein a measured amount of binding between the monoclonal antibody and the C-terminal biomarker of 1.7ng/mL or greater indicates that the patient has or is likely to develop fibrostenosis.
8. An assay kit comprising a monoclonal antibody specifically reactive with a C-terminal biomarker having the amino acid sequence PMKTMKGPFG (SEQ ID NO:1) and at least one of:
-a streptavidin coated well plate;
biotinylated peptide Biotin-L-PMKTMKGPFG (SEQ ID NO:4), wherein L is an optional linker;
-a secondary antibody for use in a sandwich immunoassay;
-a calibration peptide comprising sequence PMKTMKGPFG;
-an antibody biotinylation kit;
antibody HRP labeling kit;
-an antibody radiolabelling kit;
assay visualization kit.
9. The assay kit of claim 8, wherein a monoclonal antibody directed against a synthetic peptide having amino acid sequence PMKTMKGPFG (SEQ ID NO:1) is raised.
10. The assay kit according to claim 8 or 9, wherein the kit is for diagnosing ulcerative colitis or colorectal cancer, or for identifying patients with crohn's disease who have or may develop stenosis.
11. An immunoassay method for diagnosing, and/or monitoring, and/or assessing the likelihood of colorectal cancer or ulcerative colitis in a patient, the method comprising contacting a sample of biological fluid obtained from the patient with an antibody reactive with type XVI collagen, or a fragment thereof, thereby determining the amount of binding between the antibody and the type XVI collagen, or a fragment thereof, and correlating the amount of binding with a value associated with a normal healthy subject, and/or a value associated with a known disease severity, and/or a value obtained from the patient at a previous point in time, and/or a predetermined statistical cut-off value.
12. The method of claim 11, wherein the detecting is quantitative.
13. The method of claim 11 or 12, wherein the antibody specifically reacts with a C-terminal biomarker having a C-terminal amino acid sequence PMKTMKGPFG (SEQ ID NO: 1).
14. The method of claim 13, wherein the antibody is a monoclonal antibody.
15. The method of claim 11 or 12, wherein the antibody does not specifically recognize or bind a C-extended version of the C-terminal amino acid sequence or a C-truncated altered version of the C-terminal amino acid sequence.
16. The method of claim 14, wherein the statistical cutoff for the amount of binding between the monoclonal antibody and the C-terminal biomarker is at least 1.0 ng/mL.
17. The method of claims 11-16, wherein the biological fluid sample is blood, urine, synovial fluid, serum, or plasma.
18. An immunoassay method of diagnosing the presence of or assessing the likelihood of development of a fibrous stenosis in a patient having crohn's disease, the method comprising contacting a biological fluid sample obtained from the patient with a monoclonal antibody specifically reactive with a C-terminal biomarker having a C-terminal amino acid sequence PMKTMKGPFG (SEQ ID NO:1), and determining an amount of binding between the monoclonal antibody and the biomarker, wherein a determined amount of binding of 1.7ng/mL or more indicates the presence of or the likelihood of development of a fibrous stenosis in the patient.
19. The method of claim 18, wherein the monoclonal antibody does not specifically recognize or bind a C-extended version of the C-terminal amino acid sequence or a C-truncated shortened version of the C-terminal amino acid sequence.
20. The method of claim 18 or 19, wherein the biological fluid sample is blood, urine, synovial fluid, or plasma.
21. A monoclonal antibody specifically reactive with a C-terminal biomarker having the amino acid sequence PMKTMKGPFG (SEQ ID NO: 1).
22. A cell line that produces the monoclonal antibody of claim 21.
Technical Field
The present invention relates to a collagen type XVI assay and its use in the assessment of diseases associated with collagen type XVI, in particular colorectal cancer and ulcerative colitis, and for identifying a subgroup of crohn's disease patients suffering from (or likely to develop) fibrostenosis.
Background
Extracellular matrix (ECM) is a non-cellular component responsible for maintaining tissue architecture. Altered ECM remodeling is an important part of the pathology of Gastrointestinal (GI) diseases, such as colorectal cancer (CRC) (1) and Ulcerative Colitis (UC) (2). An imbalance between ECM formation and colonic degradation can lead to changes in ECM composition, leading to tissue dysfunction. Increased deposition of ECM proteins in the tumor microenvironment increases ECM rigidity, thereby affecting cellular functions such as cell proliferation, adhesion, migration and invasion (3, 4). It has also been evident that inflammatory responses in the tumor microenvironment affect ECM remodeling (5, 6). Also, in UC, the ECM of the intestinal tract is severely affected by chronic inflammation, which results in loss of tissue homeostasis and an imbalance in collagen metabolism (2, 7-9). Chronic inflammation and continued renewal of epithelial cells lead to dysplasia, which may be converted to CRC (10). Thus, biomarkers reflecting this enhanced ECM remodeling may be important for identifying patients with tissue/ECM architecture disruption leading to the development and progression of CRC and UC.
In the intestinal tract, type XVI collagen (hereinafter referred to as col-16) is thought to contribute to stabilization and maintenance of basement membrane, which is an ECM-specific layer located below epithelial and endothelial cell layers (11). Col-16 is a fiber-associated collagen protein, has an interrupted triple helix (FACIT), and is expressed by epithelial cells and subepithelial myofibroblasts. They underlie the basement membrane and col-16 is clearly deposited in the stroma of the epithelial crypt (11). Skin studies have shown that col-16 is located at the dermal-epidermal junction near the basement membrane, which suggests that col-16 plays a positive role in anchoring the microfibrils to the basement membrane (12, 13).
Col-16 interacts with α 1 β 1 and
An increase in col-16 expression was also detected in subepithelial fibroblasts isolated from inflamed Crohn's disease tissue biopsies (11).
The inventors have now established a pathological link between col-16 and UC and CRC, and have also developed a method for identifying a subset of crohn's disease patients who have (or are likely to develop) stenosis of the fiber.
Summary of The Invention
Accordingly, in a first aspect, the present invention relates to a method of detecting type XVI collagen, or a fragment thereof, in a sample of human biological fluid, said method comprising:
a) obtaining a biological fluid sample from a human patient; and
b) contacting the biological fluid sample with a peptide having the C-terminal amino acid sequence PMKTMKGPFG (SEQ ID NO:1) and detecting binding between the biomarker and the antibody.
The detection is preferably quantitative.
Preferably, monoclonal antibodies directed against synthetic peptides having amino acid sequence PMKTMKGPFG (SEQ ID NO:1) are raised. The synthetic peptide may be linked to a carrier protein such as, but not limited to, Keyhole Limpet Hemocyanin (KLH).
In a preferred embodiment, the monoclonal antibody does not specifically recognize or bind to a C-extended version of the C-terminal amino acid sequence or a C-truncated shortened version of the C-terminal amino acid sequence. In this regard, "a C-extended version of the C-terminal amino acid sequence" refers to one or more amino acids that extend beyond the C-terminus of the sequence PMKTMKGPFG-COOH (SEQ ID NO: 1). For example, if the C-terminal amino acid sequence PMKTMKGPFG-COOH (SEQ ID NO:1) is extended by a glycine residue, the corresponding "extended version of the C-extension" would be PMKTMKGPFGG-COOH (SEQ ID NO: 2). Similarly, it is preferred that the antibody does not specifically recognize or bind to a C-truncated shortened version of the C-terminal amino acid sequence. In this regard, "a C-truncated shortened version of the C-terminal amino acid sequence" refers to one or more amino acids removed from the C-terminus of the sequence PMKTMKGPFG-COOH (SEQ ID NO: 1). For example, if the C-terminal amino acid sequence PMKTMKGPFG-COOH (SEQ ID NO:1) is shortened by one amino acid residue, the corresponding "C-truncated shortened version" would be PMKTMKGPF-COOH (SEQ ID NO: 3).
By using various statistical analyses, it was found that a measured value of the amount of binding between the monoclonal antibody (as described above) and the C-terminal biomarker of 1.0ng/mL or more was highly correlated with the likelihood of ulcerative colitis or colorectal cancer. In this regard, it was found that in the total population being screened (including healthy subjects, patients with UC and patients with CRC), at least 90% of the subjects in this population had a C-terminal biomarker level of ≧ 1.0ng/mL with UC or CRC. Thus, by setting the cut-off value to 1.0ng/mL, the method of the present invention can be used to predict the likelihood of ulcerative colitis or colorectal cancer with high confidence. Or, in other words, applying a statistical cut-off to the method of the invention is particularly advantageous, since it results in an independent predictive determination; that is, it eliminates the need to make any direct comparison to healthy individuals and/or patients of known disease severity to reach a diagnostic conclusion. This may also be particularly advantageous when using the assay to assess a patient who already has medical signs or symptoms that are generally indicative of colorectal cancer or ulcerative colitis (e.g., as determined by physical examination and/or negotiation with a medical professional), as it may serve as a quick and definitive tool to confirm the initial prognosis, thus potentially eliminating the need for more invasive procedures (e.g., endoscopy) and speeding up the start of an appropriate treatment regimen. In the special case of colorectal cancer, a rapid conclusive diagnosis may lead to the disease being discovered and treated at an earlier stage, which in turn may improve the overall chances of survival. Thus, a statistical cutoff value may be used to assess the risk of a patient suffering from or developing UC or CRC.
Furthermore, in the case of patients with Crohn's disease, it has been found that a measured amount of binding between the monoclonal antibody (as described above) and the C-terminal biomarker of 1.7ng/mL (statistical cut-off) or higher correlates well. The likelihood that the patient has or may develop a stenosis that is a fibrous stenosis. This is advantageous for the same reasons as described above.
Thus, in a second aspect, the present invention relates to an immunoassay method for diagnosing, and/or monitoring, and/or assessing the likelihood of colorectal cancer or ulcerative colitis in a patient, the method comprising contacting a sample of biological fluid obtained from the patient with an antibody reactive with collagen type XVI, or a fragment thereof, thereby determining the amount of binding between the antibody and the collagen type XVI, or fragment thereof, and correlating the amount of binding with a value associated with a normal healthy subject, and/or a value associated with a known disease severity, and/or a value obtained from the patient at a previous point in time, and/or a predetermined statistical cut-off value.
In particular, the methods may be used to monitor the progression of UC or CRC in a patient and/or the effect of treatment on patients suffering from UC or CRC. For example, the first value may be obtained at a first time point before the start of the treatment and the second value may be obtained at a second later time point after the start of the treatment. A decrease in the amount of binding measured by the method from the first time point to the second time point would indicate an improvement in the patient's condition, thus indicating that the patient is responsive to treatment. Conversely, an increase in the amount of binding measured by the method from the first time point to the second time point will indicate a worsening of the patient's condition, thus indicating that the patient is not responding to the treatment. Thus, the methods may be used to monitor and/or assess the efficacy of novel therapies, such as, but not limited to, new drugs or antibody therapies.
Similarly, in the case of a patient having Crohn's disease, the present invention relates to an immunoassay method for diagnosing the presence of or assessing the likelihood of developing a stenosis of a fibrous stenosis in a patient having Crohn's disease, the method comprising contacting a sample of biological fluid obtained from said patient with an antibody specifically reactive with a C-terminal biomarker having C-terminal amino acid sequence PMKTMKGPFG (SEQ ID NO:1), and determining the amount of binding between said antibody and said biomarker, wherein a determined amount of binding of 1.7ng/mL or more indicates the presence of a stenosis or the likelihood of developing a fibrous stenosis in said patient. Also, this method is advantageous for the same reasons as described above (monitoring of the progress of the disease, therapeutic effect, etc.).
The detection is preferably quantitative. The fragment is preferably a C-terminal fragment of collagen XVI.
Preferably, the antibody specifically reacts with a C-terminal biomarker having the amino acid sequence PMKTMKGPFG (SEQ ID NO: 1). More preferably, the antibody is a monoclonal antibody. Preferably, the antibody does not specifically recognize or bind to the C-extended version of the C-terminal amino acid sequence or the C-truncated altered version of the C-terminal amino acid sequence.
For the reasons described above, the immunoassay of the second aspect may utilize a statistical cut-off of 1.0ng/mL for the C-terminal biomarker to determine the likelihood of UC or CRC.
In any of the methods of the invention described herein, the biological fluid sample can be, but is not limited to, blood, urine, synovial fluid, serum, or plasma.
In any of the methods of the invention described herein, the method of immunoassay may be, but is not limited to, a competition assay or a sandwich assay. The method of immunoassay may be, but is not limited to, radioimmunoassay or enzyme-linked immunosorbent assay.
In a third aspect, the present invention relates to an assay kit comprising a monoclonal antibody specifically reactive with a C-terminal biomarker having the amino acid sequence PMKTMKGPFG (SEQ ID NO:1) and at least one of:
a streptavidin coated well plate;
biotinylated peptide Biotin-L-PMKTMKGPFG (SEQ ID NO:4), where L is an optional linker;
a secondary antibody for use in a sandwich immunoassay;
a calibration peptide-COOH (SEQ ID NO:1) comprising sequence PMKTMKGPFG;
antibody biotinylation kits;
antibody HRP labeling kit;
antibody radiolabelling kits;
and (4) a visual determination kit.
The kit may be used for diagnosing or predicting the risk of ulcerative colitis or colorectal cancer, or for identifying patients with crohn's disease who have or are likely to develop a fibrostenotic stenosis disease phenotype, preferably in combination with any of the above methods.
Definition of
As used herein, the term "C-terminal" refers to the end of a polypeptide, i.e., at the C-terminus of a polypeptide, and should not be construed as meaning in its general direction.
As used herein, the term "competitive ELISA" refers to a competitive enzyme-linked immunosorbent assay and is a technique known to those skilled in the art.
As used herein, the term "sandwich immunoassay" refers to the use of at least two antibodies for detecting an antigen in a sample and is a technique known to those skilled in the art.
As used herein, the term "amount of binding" refers to a quantification of the binding between an antibody and a biomarker, the quantification being determined by comparing a measured value of the biomarker in a biological fluid sample to a calibration curve, wherein the calibration curve is generated using a standard sample of known concentrations of the biomarker. In the particular assay disclosed herein that measures C-terminal biomarkers having the C-terminal amino acid sequence PMKTMKGPFG (SEQ ID NO:1) in biological fluids, a calibration curve was generated using a standard sample of known concentration of calibration peptide PMKTMKGPFG (SEQ ID NO: 1). The measured values in the biological fluid sample are compared to a calibration curve to determine the actual amount of the biomarker in the sample. The invention uses spectrophotometry to analyze and generate a standard curve and measure the binding capacity in the biological fluid sample; in the examples listed below, the method utilizes HRP and TMB to produce a measurable color intensity that is proportional to the amount of binding and is readable by a spectrophotometer. Of course, any other suitable analysis method may be used.
As used herein, "statistical cut-off" refers to a statistically determined amount of binding that indicates a high likelihood of UC or CRC in a patient, or that indicates a Crohn's Disease (CD) patient with (or likely to develop) fibrostenosis, because a measurement of a biomarker in a patient sample (preferably a serum sample) that is equal to or higher than the statistical cut-off corresponds to a probability of at least 80%, preferably a probability of at least 85-6, more preferably a probability of at least 90%, most preferably a probability of at least 95% for the presence of UC or CRC, or (for CD patients) of stenosis or likelihood of the presence or appearance of fibrostenosis.
As used herein, the term "a value associated with a normal healthy subject and/or a value associated with a known disease severity" refers to a standardized amount of type XVI collagen determined by the above methods for a subject considered healthy (i.e., without UC or CRC), and/or a standardized amount or standard amount of type XVI collagen determined by the above methods for a subject known to have UC or CRC of known severity.
Brief Description of Drawings
FIG. 1: specificity of the PRO-C16 assay (also referred to as "C16-C" in the figure). % B/B0: b is equal to OD at x nM peptide, B0 is equal to OD at 0nM peptide.
FIG. 2: serum PRO-C16 levels in patients with colorectal cancer (CRC) and Ulcerative Colitis (UC) compared to healthy controls; (a) levels of PRO-C16 in serum from controls (n-50), CRC (n-50) and UC patients (n-39). Levels below the lower limit of the measurement range (LLMR) will be adjusted to LLMR. Results are shown in Tukey box diagrams. These boxes represent the 25 th and 75 th percentiles of the median. Whiskers represent the lowest and highest values, except for the outliers (. cndot.), which are 1.5 times higher than the 75 th percentile. Groups were compared using the Kruskal Wallis test. Asterisks indicate the following: p <0.01 and p < 0.0001; (b) the level of PRO-C16 in sera from CRC patients, UC patients, and controls was divided by the quartile (Q). The number of control, CRC and UC patients in each group is indicated. The cut-off value (1.0ng/mL) obtained from the ROC curve is indicated by a dotted line; (c) PRO-C16 levels in serum of CRC patients were compared at baseline and three months after tumor resection (follow-up). Statistically significant differences were determined using the paired Wilcoxon test. p > 0.9999.
FIG. 3 evaluation of PRO-C16 in serum of colorectal cancer (CRC) patients based on tumor stage. The level of PRO-C16 in serum from CRC patients at baseline was divided into disease stages, with the median indicated by the horizontal line. Groups were compared using the Kruskal-Wallis test.
FIG. 4 evaluation of PRO-C16 in serum of Crohn's disease patients. B1: CD patients with luminal disease; b2: CD patients with a fibrostenotic stenosis disease phenotype; b3: CD patients with a fistulopathy phenotype.
Detailed Description
Examples
Embodiments of the present disclosure are described in the following examples, which are intended to aid in understanding the disclosure, and should not be construed to limit in any way the scope of the disclosure as defined in the claims that follow thereafter. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the disclosure nor are they intended to represent that the experiments below are all or only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.
In the following examples, the following materials and methods were employed.
Material
All reagents used in the experiments were standard chemicals from merck (white hause station, new jersey) and sigma aldrich (st louis, missouri). Synthetic peptides for antibody production and assay development were purchased from chinese polypeptide corporation (beijing, china) (table 1).
Selection and overview of peptides
Epitope selection
The C-terminus of the I chain of col-16 was selected as the target epitope and is referred to herein as "PRO-C16". The C-terminal PRO-C16 amino acid sequence 1595' -PMKTMKGPFG (SEQ ID NO:1) was used to generate antibodies specific for the C-terminus of col-16. In addition, it was used to design a selection peptide (PMKTMKGPFG; SEQ ID NO:1) (Table 1). Using the Uniprot/Swiss-Prot database (20) using NPS @: network protein sequence analysis, sequences were BLASTed to be homologous to other human proteins and species. The amino acid sequence is unique to human col-16. Streptavidin-coated plates used in the ELISA were coated with biotinylated peptide (Biotin-K-PMKTMKGPFG). Elongated peptide (PMKTMKGPFGG; SEQ ID NO: 2), truncated peptide (PMKTMKGPF; SEQ ID NO: 3), nonsense peptide (VPKDLPPDTT; SEQ ID NO: 5) and nonsense biotinylated peptide (Biotin-VPKDLPPDTT; SEQ ID NO: 3) NO: 6) are included to test the specificity of the antibody.
TABLE 1 synthetic peptides for antibody production and assay development
KLH, keyhole limpet hemocyanin
Monoclonal antibody production and clonal characterization
Production of monoclonal antibodies was performed as described previously (21). Briefly, 4 to 6 week old Balb/C mice were immunized subcutaneously with 200. mu.l of emulsified antigen and 50. mu.g of immunogenic peptide (Keyhole Lippetehemocyanin (KLH) -CGG-PMKTMKGPFG; SEQ ID NO: 7) using Freund's incomplete adjuvant (Sigma-Aldrich, St. Louis, Mo., U.S.A.). Mice were immunized at two week intervals until stable serum titer levels were reached. Mice with the highest serum titers were selected for fusion, left for one month, and then immunized intravenously with 50 μ g of the immunogenic peptide in 100 μ l of 0.9% NaCl solution. Three days later, splenocytes were isolated for cell fusion. Briefly, splenocytes were fused with SP2/0 myeloma cells to produce hybridoma cells, which were then cloned into culture dishes using the half-medium method (22). Clones were seeded into 96-well microtiter plates and limited dilutions were used to ensure monoclonal growth. The supernatants were screened for reactivity to the selection peptide (PMKTMKGPFG; SEQ ID NO:1) and native material (serum) in an indirect competition ELISA using streptavidin-coated plates (Roche, Hvidovre, Denmark, cat.11940279). The clones with the best reactivity were purified using a protein G-column according to the manufacturer's instructions (GE Healthcare Life Sciences, LittleChalfount, Buckinghamshire, UK). Two monoclonal pairs were tested to select for peptide (PMKTMKGPFG; SEQ ID NO:1) rather than extended (PMKTMKGPFGG; SEQ ID NO: 2), truncated (PMKTMKGPF; SEQ ID NO: 3) or nonsense peptide (VPKDLPPDTT; SEQ ID NO: 5). One monoclonal was selected for assay development. The optimal incubation buffer, time, temperature and optimal ratio of biotinylated peptide to antibody were determined.
PRO-C16ELISA protocol
The PRO-C16 competitive ELISA procedure was as follows: a96-well streptavidin-coated microtiter plate was treated with 100. mu.l biotinylated peptide (Biotin-K-PMKTMKGPFG; SEQ ID NO:4) (final concentration 3.1.0ng/mL) in assay buffer (50mM PBS-BTB, 4g/l NaCl, pH 7.4). The plate was incubated at 20 ℃ for 30 minutes with shaking (300rpm) and then washed five times in wash buffer (20mM TRIS, 50mM NaCl, pH 7.2). A volume of 20. mu.l of sample/control/selection peptide (PMKTMKGPFG; SEQ ID NO:1) was added, followed immediately by 100. mu.l of monoclonal antibody (final concentration 62.5ng/ml) diluted in assay buffer. The plates were incubated at 20 ℃ for 1 hour with shaking and then washed five times in wash buffer. Then, 100. mu.l of goat anti-mouse HRP-conjugated IgG antibody (Thermo Scientific, Waltham, MA, USA; catalog #31437) diluted in assay buffer (final concentration of 130ng/ml) was added to eachIn the hole. The plates were incubated at 20 ℃ for 1 hour with shaking, followed by five washes in wash buffer. Next, 100. mu.l of tetramethylbenzidine (TMB, Kem-En-Tec Diagnostics, Taastrup, Denmark) was added and incubated at 20 ℃ for 15 minutes in the dark with shaking. To stop the reaction of TMB, 100. mu.l of 1% H was added2SO4And the plate was analysed at 450nm with 650nm as reference in a VersaMax ELISA microplate reader. Standard curves were drawn using a 4-parameter mathematical fit model and data were analyzed using Softmax Pro v.6.3 software.
Technical assessment
Antibody specificity was calculated as the percentage of signal inhibition for the 2-fold dilution of the selection peptide (PMKTMKGPFG; SEQ ID NO:1), extension peptide (PMKTMKGPFGG; SEQ ID NO: 2), truncated peptide (PMKTMKGPF; SEQ ID NO: 3) or nonsense peptide (VPKDLPPDTT; SEQ ID NO: 5). The lower limit of the measurement range (LLMR) and the upper limit of the measurement range (ULMR) were calculated from the standard curves of 10 independent runs. A 2-fold dilution of a healthy serum sample from humans (n-3) was used to determine linearity and was calculated as recovery of the undiluted sample. Seven samples covering the detection range of the PRO-C16 analysis (LLMR-ULMR) were run ten times independently to calculate the difference between the internal analysis and the internal analysis. The seven samples included three human serum samples and four selected peptide samples spiked with assay buffer. The intra-assay differences were determined as the mean coefficient of variation (CV%) between plates, and the inter-assay differences were calculated as the mean CV% between plates. Accuracy was determined from three human serum samples spiked with two-fold dilutions of the selected peptide and calculated as percent recovery of the expected concentration (serum and peptide bound). The analyte stability of three healthy serum samples subjected to three freeze and thaw cycles was determined. The freeze-thaw recovery was calculated with reference to the first cycle. In addition, the stability of the analyte was determined by incubating three human serum samples at 4 ℃ or 20 ℃ for 24 or 48 hours. Recovery was calculated with reference to samples stored at-20 ℃. Interference was determined by adding low/high amounts of biotin (1.5/4.5ng/ml), lipids (0.75/2.5mg/ml), or hemoglobin (1.25/2.5mg/ml) to serum samples of known concentration and calculated as percent recovery of analyte in unlabeled serum.
Patient serum sample
According to the declaration of Helsinki, serum samples from CRC patients were collected by medical personnel at the Johnger hospital Copenhagen Bisby, Denmark, after informed consent and approval by the Ethics Committee of the first metropolitan area, Denmark (Dermata, first Copenhagen; approval number: Hl-2014-048). Serum samples were collected before (baseline) and three months post-operative (follow-up) in 50 and 23 tumor resections, respectively. Tumor stage was assessed according to the international cancer control alliance classification system.
Serum samples of UC patients (n-39) were obtained from the university of european hospital, europe denmark, after informed consent. The levels of PRO-C16 in CRC and UC patients were compared to levels in commercially available control sera of healthy donors (n 50) (Valley BioMedical, Winchester, VA, USA), which documents all had informed consent based on the manufacturer's information. Information related to the included patients is shown in table 2. According to the danish law, no additional ethical approval needs to be obtained when measuring biochemical markers in previously collected samples.
TABLE 2 Main clinical characteristics of the study population
Statistical analysis
Multiple comparisons of adjusted one-way analysis of variance (ANOVA) were performed using the Kruskal-Wallis test for comparison of PRO-C16 serum levels in control, baseline CRC and UC patients. CRC patients at baseline and follow-up were compared using the Wilcoxon test. The odds ratios and positive predicted values were generated from a specific critical value (1.0ng/mL) obtained from the Receiver Operating Characteristics (ROC) curve and analyzed using Fisher's exact probability test and chi-square test. A p-value p <0.05 is considered statistically significant. Statistically significant differences are marked with asterisks in the figures and are illustrated in the legend. Prism 7 software (Graphpad v7.01) was used for all statistical analyses.
Specificity of PRO-C16 detection
The specificity of the newly developed PRO-C16ELISA was evaluated by studying the inhibitory effect of the different peptides. The peptide (PMKTMKGPFG; SEQ ID NO:1) was selected to suppress the signal to 6%, whereas only slight suppression was detected using the extension peptide (PMKTMKGPFGG; SEQ ID NO: 2), the truncated peptide (PMKTMKGPF; SEQ ID NO: 3) and the nonsense peptide (VPKDLPPDTT; SEQ ID NO: 5) and was present only at the highest concentration (FIG. 1). No reactivity to nonsense biotinylated peptide (Biotin-VPKDLPPDTT; SEQ ID NO: 6) was observed. Taken together, this indicates that the antibody is specific for the C-terminus of col-16.
Technical evaluation of PRO-C16 analysis
Several tests were included to evaluate the overall technical performance of the PRO-C16 analysis (table 3). The measurement range was determined by calculating LLMR and ULMR, which ranged from 0.87 to 95.50 ng/ml. The intra-and inter-batch differences were 10% and 15%, respectively. Natural reactivity was observed in human serum. From undiluted to 1: after 4 dilutions, the recovery of the dilutions in serum was 95%. The addition of standard peptide in human serum results in average recovery of 99%, indicating accuracy and no influence of sample matrix on determination reaction. After four freeze-thaw cycles, the stability of the analyte was acceptable with a recovery of 103%. The human serum was stored at 4 ℃ for 24 or 48 hours, and then the analyte was recovered at 106% and 95%, respectively. Recovery of 91% and 85% was recovered by storage at 20 ℃ for 24 or 48 hours, respectively. No interference was detected at either low or high levels of biotin, lipids or hemoglobin.
TABLE 3 technical validation of the PRO-C16 assay
LLMR, lower measurement Range limit; ULMR, upper limit of measurement range. The percentages are reported as mean values.
Serum PRO-C16 levels in colorectal cancer and ulcerative colitis
To determine the biomarker potential of col-16, PRO-C16 levels were measured in sera obtained from CRC and UC patients and compared to healthy controls. The levels of PRO-C16 were significantly increased in patients with CRC (p < 0.0026) and UC (p <0.0001) compared to healthy controls (fig. 2 a). With increasing quartiles, the percentage of cases of CRC and UC in the total test population increased stepwise (fig. 2 b). In the population with PRO-C16 levels in the upper quartile (Q4), patients with CRC or UC were 97% (34/35) and healthy controls were 3% (1/35). PRO-C16 was able to identify patients with CRC or UC with a positive predictive value of 0.9 and an odds ratio of 12 (95% CI: 4.5-29.5, p < 0.0001).
This indicates that levels of PRO-C16 are able to distinguish CRC and UC patients from healthy controls. Thus, PRO-C16 is often measured in serum for its biomarker potential in gastrointestinal disorders.
When PRO-C16 levels were compared (paired) in CRC patients between pre-tumor resection (baseline) and three months post-tumor resection (follow-up), no difference was observed (p >0.999) (fig. 2C). This indicates that col-16 does not originate from the primary tumor.
Since tumor staging is an important clinical tool in CRC, PRO-C16 levels were divided according to tumor stage (fig. 3). No significant differences were detected between tumor stages. However, a trend of elevated levels of PRO-C16 was observed in stages II and III.
Serum PRO-C16 levels in patients with Crohn's disease
Intestinal fibrosis is a common complication of Inflammatory Bowel Disease (IBD), but is more prevalent in crohn's disease, which can progress to fibrostenosis.
Intestinal and myofibroblasts are the major effector cells of the development of intestinal fibrosis, and intestinal subepithelial fibroblasts (ISEM) have shown a significant increase in collagen levels in XVI in CD patients (11).
Thus, various subgroups of Crohn's Disease (CD) patients were evaluated using the PRO-C16 assay:
-a CD patient with intracavity disease (B1);
-CD patients have a fibrostenotic disease phenotype (B2);
CD patients with a fistulopathy phenotype (B3).
The levels of PRO-C16 biomarker were detected in CD patients with fibrostenosis (B2) unexpectedly high (and statistically significant) compared to cohorts of CD patients with luminal disease (B1), CD patients with fistula disease phenotype (B3) and healthy donors (fig. 4).
Thus, these results indicate that the PRO-C16 assay can be used to diagnose CD patients with stenosis, or stenosis that may develop, fibrostenosis. This is a significant finding, as recent reviews of narrow crohn's disease assessments (23, 24) indicate that there is a continuing need for non-invasive methods to assess stenosis. These reviews also indicate that there are currently no serological biomarkers that can reliably predict the risk of developing intestinal stenosis or identify early stages of fibrosis before clinical symptoms appear; candidate biomarkers of intestinal fibrosis have not been shown to have strict specificity for fibrostenosis. Thus, there remains a need for such specific serological biomarkers for non-invasive assessment of CD patients having (or likely to develop) a fibrostenotic stenosis disease phenotype.
Thus, a statistically significant increase in the level of the PRO-C16 biomarker, as identified in the B2 cohort, indicates that the PRO-C16 assay can be used to reliably identify CD patients who have (or are likely to develop) fibrostenosis. In this regard, a measured PRO-C16 value of at least 1.7ng/mL (statistical cutoff), preferably at least 2.0ng/mL, for samples obtained from CD patients is considered indicative of CD patients having (or likely to develop) fibrous stenosis.
Conclusion
A reliable competitive ELISA has been developed and validated that is capable of non-invasively measuring col-16 (PRO-C16). Using the PRO-C16 assay described herein, a significant increase in the level of PRO-C16 in serum from CRC and UC patients was observed compared to healthy controls. To our knowledge, this is the first study showing a link between Col-16 and UC or CRC, and we predict that pathological links may exist between Col-16 and other diseases (e.g. melanoma). In addition, the PRO-C16ELISA showed specificity for CD patients with (or likely to develop) a fibrostenotic stenosis disease phenotype, a significant improvement over current serological biomarkers for this purpose.
In this specification, unless explicitly stated otherwise, the use of the word "or" refers to an operator that returns a true value when two or more of the stated conditions are met, as opposed to the operator "exclusive or", which need only meet one of the following conditions. The term "comprising" means "including" rather than "consisting of. All prior teachings acknowledged above are incorporated herein by reference. Any prior published document in this document should not be taken as an acknowledgement or admission that the teachings thereof were common general knowledge in australia or elsewhere on the date of the present report.
Reference to the literature
1.Kehlet SN,Sanz-Pamplona R,Brix S,Leeming DJ,Karsdal MA,MorenoV.Excessive collagen turnover products are released during colorectal cancerprogression and elevated in serum from metastatic colorectal cancerpatients.Sci Rep[Internet].2016；6(1):30599.Available from:http://www.nature.com/articles/srep30599
2.Lawrance IC,Maxwell L,Doe W.Inflammation location,but not type,determines the increase in TGF-betal and IGF-1 expression and collagendeposition in IBD intestine.Inflamm Bowel Dis[Internet].2001；7(1):16-26.Available from:http://www.ncbi.nlm.nih.gov/pubmed/11233656
3.Provenzano PP,Eliceiri KW,Campbell JM,Inman DR,White JG,KeelyPJ.Collagen reorganization at the tumor-stromal interface facilitates localinvasion.BMC Med[Internet].2006；4(1):38.Available from:http://bmcmedicine.biomedcentral.com/articles/10.1186/1741-7015-4-38
4.Egeblad M,Rasch MG,Weaver VM.Dynamic interplay between the collagenscaffold and tumor evolution.Vol.22,Current Opinion in CellBiology.2010.p.697-706.
5.Iijima J,Konno K,Itano N.Inflammatory alterations of theextracellular matrix in the tumor microenvironment.Vol.3,Cancers.2011.p.3189-205.
6.van Kempen LCL,de Visser KE,Coussens LM.Inflammation,proteases andcancer.Eur J Cancer.2006；42(6):728-34.
7.Ravi A,Garg P,Sitaraman S V.Matrix metalloproteinases ininflammatory bowel disease:Boon or a bane？Vol.13,Inflammatory BowelDiseases.2007.p.97-107.
8.Shimshoni E,Yablecovitch D,Baram L,Dotan I,Sagi I.ECM remodellingin IBD:innocent bystander or partner in crime？The emerging role ofextracellular molecular events in sustaining intestinal inflammation.Gut[Internet].2015；64(3):367-72.Available from:http://gut.bmj.com/lookup/doi/10.1136/gutjnl-2014-308048
9.Mortensen JH 0g,Godskesen LE lbjerg,Jensen MD am,Van Haaften WTobias,Klinge LG abriels,Olinga P,et al.Fragments of Citrullinated and MMP-degraded Vimentin and MMP-degraded Type III Collagen Are Novel SerologicalBiomarkers to Differentiate Crohn's Disease from Ulcerative Colitis.J CrohnsColitis.2015；9(10):863-72.
10.Rogler G.Chronic ulcerative colitis and colorectal cancer.Vol.345,Cancer Letters.2014.p.235-41.
11.Ratzinger S,Eble JA,Pasoldt A,Opolka A,Rogler G,Grifka J,etal.Collagen XVI induces formation of focal contacts on intestinalmyofibroblasts isolated from the normal and inflamed intestinal tract.MatrixBiol.2010；29(3):177-93.
12.Grassel S,Unsold C,Schacke H,Bruckner-Tuderman L,BrucknerP.Collagen XVI is expressed by human dermal fibroblasts and keratinocytes andis associated with the microfibrillar apparatus in the upper papillarydermis.Matrix Biol.1999；18(3):309-17.
13.Kassner A,Hansen U,Miosge N,Reinhardt DP,Aigner T,Bruckner-Tuderman L,et al.Discrete integration of collagen XVI into tissue-specificcollagen fibrils or beaded microfibrils.Matrix Biol.2003；22(2):131-43.
14.Grassel S,Bauer RJ.Collagen XVI in health and disease.Vol.32,Matrix Biology.2013.p.64-73.
15.Eble JA,Kassner A,Niland S,Morgelin M,Grifka J,Grassel S.CollagenXVI harbors an integrinrecognition site in its C-terminal domains.J BiolChem.2006；281(35):25745-56.
16.Bedal KB,Gr？？ssel S,Oefner PJ,Reinders J,Reichert TE,BauerR.Collagen XVI induces expression of MMP9 via modulation of AP-1transcription factors and facilitates invasion of oral squamous cellcarcinoma.PLoS One.2014；9(1).
17.Ratzinger S,Grassel S,Dowejko A,Reichert TE,Bauer RJ.Induction oftype XVI collagen expression facilitates proliferation of oral cancercells.Matrix Biol.2011；30(2):118-25.
18.Bauer R,Ratzinger S,Wales L,Bosserhoff A,Senner V,Grifka J,etal.Inhibition of collagen XVI expression reduces glioma cellinvasiveness.Cell Physiol Biochem.2011；27(3-4):217-26.
19.Senner V,Ratzinger S,Mertsch S,Grassel S,Paulus W.Collagen XVIexpression is upregulated in glioblastomas and promotes tumor celladhesion.FEBS Lett.2008；582(23-24):3293-300.
20.Combet C,Blanchet C,Geourjon C,Deleage [email protected]:network proteinsequence analysis.Trends Biochem Sci.2000 Mar；25(3):147-50.
21.Nielsen MJ,Nedergaard AF,Sun S,Veidal SS,Larsen L,Zheng Q,etal.The neo-epitope specific PRO-C3 ELISA measures true formation of type IIIcollagen associated with liver and muscle parameters.Am J Transl Res.2013；5(3):303-15.
22.Gefter ML,Margulies DH,Scharff MD.A simple method for polyethyleneglycol-promoted hybridization of mouse myeloma cells.Somatic Cell Genet.1977；3(2):231-6.
23.Giuffrida P,Pinzani M,Corazza GR,Sabatino A,Biomarkers ofintestinal fibrosis-one step towards clinical trials for stricturinginflammatory bowel disease.United European Gastroenterol J.2016 Aug；4(4):523-530.
24.Bettenworth D,Nowacki TM,Cordes D,Buerke B,Lenze F,Assessment ofstricturing Crohn's disease:Current clinical practice and futureavenues.World J Gastroenterol.2016 Jan 21；22(3):1008-1016.
Sequence listing
<110> North European Biotechnology Co
<120> type XVI collagen assay
<130>P20115WO
<150>GB1717301.4
<151>2017-10-20
<160>7
<170>BiSSAP 1.3.6
<210>1
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>
<222>
<223> C-terminal biomarker (C-term biomarker)
<400>1
Pro Met Lys Thr Met Lys Gly Pro Phe Gly
1 5 10
<210>2
<211>11
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>
<222>
<223> extended peptide (elongated peptide)
<400>2
Pro Met Lys Thr Met Lys Gly Pro Phe Gly Gly
1 5 10
<210>3
<211>9
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>
<222>
<223> Truncated peptide (Truncated peptide)
<400>3
Pro Met Lys Thr Met Lys Gly Pro Phe
1 5
<210>4
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>SITE
<222>1
<223> biotinylation with an optional linker between biotin and peptide
<220>
<221>
<222>
<223> biotinylated peptide with optional linker
<400>4
Pro Met Lys Thr Met Lys Gly Pro Phe Gly
1 5 10
<210>5
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>
<222>
<223> nonsense peptide (nonsense peptide)
<400>5
Val Pro Lys Asp Leu Pro Pro Asp Thr Thr
1 5 10
<210>6
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>SITE
<222>1
<223> biotinylation with an optional linker between the peptide and biotin (Biotinylated, with optional linker between peptide and biotin)
<220>
<221>
<222>
<223> biotinylated nonsense peptide (biotinylated nonsense peptide)
<400>6
Val Pro Lys Asp Leu Pro Pro Asp Thr Thr
1 5 10
<210>7
<211>13
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>SITE
<222>1
<223> attached KLH (KLH attached)
<220>
<221>
<222>
<223> immunogenic peptide (immunogenic peptide)
<400>7
Cys Gly Gly Pro Met Lys Thr Met Lys Gly Pro Phe Gly
1 5 10