KR20180104648A - Combined biomarker assay for fibrosis - Google Patents

Abstract

The present invention relates to a cross-linked PIIINP comprising two or more PIIINP strands, each having a C-terminal neo-epitope of PIIINP formed by cleavage of intact < RTI ID = 0.0 > The present invention provides a sandwich immunoassay method for detecting a sample. Comprising contacting a biological sample comprising a crosslinked PIIINP with a first monoclonal antibody bound to a surface and then contacting the second monoclonal antibody and measuring binding of the second monoclonal antibody, And specifically reacts to the neo-epitope of the C-terminal sequence of PIIINP. The present invention also provides a method for evaluating the efficacy of an antagonist drug targeting lysyl oxidase through immunoassay and a kit comprising a solid support and a second monoclonal antibody bound to a first monoclonal antibody.

Description

Combined biomarker assay for fibrosis

The present invention relates to a sandwich immunoassay method for detecting PIIINP cross-linked in a biological sample and a method for evaluating the efficacy of a drug targeting lysyl oxidase (LOX) using the same. The present invention also relates to a kit for performing sandwich immunoassay.

Fibrosis (including the diseases listed in Table 1) is a major factor in increasing morbidity and mortality, for example, with cirrhosis worldwide reaching 800,000 deaths annually worldwide (1).

Table 1. Various fibrotic diseases (2).

group Examples of causative diseases liver Viral hepatitis
Schistosomiasis
Fatty liver (alcoholic or non-alcoholic) lungs Idiopathic pulmonary fibrosis (IPF)
Systemic sclerosis (scleroderma) kidney Shinseong Systemic Fibrosis (NSF)
diabetes
Non-therapeutic Hypertension Heart heart attack
High blood pressure
Atherosclerosis
Restenosis Eye Macular degeneration, retinitis and vitreoretinopathy skin Systemic sclerosis and scleroderma, keloids, hypertrophic scars, burns, hereditary factors
NFS Pancreas Autoimmune / hereditary factors chapter Crohn's disease / inflammatory bowel disease brain Alzheimer's disease, AIDS marrow Cancer, aging Bundle-organs fibrosis Surgical complications, chemotherapeutic agents-induced fibrosis, radio-induced fibrosis, mechanical damage

'Fibrosis disease' is any disease that causes fibrosis as a main symptom or as an adverse symptom. Fibrosis is the end result of a chronic inflammatory response caused by various stimuli including persistent infection, autoimmune reaction, allergic reaction, chemical insult, radiation and tissue damage. Fibrosis is characterized by the accumulation and reorganization of extracellular matrix (ECM). Despite the obvious pathological and clinical differences, most chronic fibrosing disorders commonly have a sustained stimulus source that sustains the production of growth factors, proteolytic enzymes, angiogenic factors, and fibrosing cytokines, and these stimuli The circles also stimulate the deposition of connective tissue factors, particularly collagen and proteoglycans, to gradually remodel and destroy normal tissue structures (3,4). Despite the considerable impact of fibrosis on human health, therapeutic agents that directly target the fibrogenic mechanism have not yet been approved (5).

Extracellular  Substrate (ECM)

ECM is a supramolecular structure with the ability to form aggregates of proteins, forming a dynamic scaffold that links cells to a three-dimensional network. This scaffold regulates cell-substrate interactions and cell fate through up- and down-regulation of proteases (6). The ECM is composed of various contents and combinations of collagen, laminin, proteoglycans and other glycoproteins, and provides various biological components, which are used to build scaffolds with specific functions to meet the needs of the individual organization It can be modified by proteases (7).

Collagen types I and III are the major structural proteins of the human body. Collagen type III is essential for collagen type I fibril formation in cardiovascular and other organs (8,9). During assembly of the fibrils, the N-terminal propeptide of type III procollagen (consisting of three identical alpha -chains, with a total molecular weight of 42 kDa) is bound to the specific N-protease before the mature collagen is incorporated into the ECM . The cleaved propeptide may be retained in the ECM or released into the circulatory system. However, cleavage of the propeptide is sometimes incomplete, leaving the propeptide attached to the molecule. As a result, abnormally crosslinked thin filaments are formed, which causes abnormal molecules to undergo rapid metabolic conversion (10, 11). Thus, in appropriate samples, the level of the N-terminal propeptide of type III collagen may be a marker for the formation and / or degradation of collagen type III.

ECM remodeling plays an important role in the development of various diseases because the modified constituents and non-coding variants of ECM cause changes in tissue stiffness and signaling potential of intact ECM and its fragments. ECM remodeling is an important prerequisite for tissue function and recovery and is tightly regulated by enzymes that cause synthesis and degradation of ECM.

When a disease such as a fibrotic disease occurs, the balance between the formation and degradation of the ECM is destroyed, and the composition of the ECM is changed. These changes change the function of the organization (12, 13). PIIINP can be used as a biomarker for several types of fibrotic diseases such as lung injury 14, viral and non-viral hepatitis 15, systemic sclerosis 16, vascular remodeling 17 and kidney disease 18 ≪ / RTI >

There was limited interest in ECM remodeling in skeletal muscle tissue. In the rat model, increased collagen gene expression and biosynthesis were identified in the quadriceps muscle and tibialis muscle after exercise (19,20). In addition, serum PIIINP levels were elevated in post-exercise clinical trials (21). That is, remodeling of skeletal muscle protein increases the amount of PIIINP in the circulatory system, so that it can be used as a biomarker for detecting premature muscle assimilation. Serum PIIINP levels have been previously proposed as biomarkers of muscle tissue response to testosterone (22), recombinant human growth hormone (23), or a combination thereof (24, 25).

In liver fibrosis, fibrillar collagen types I and III are highly upregulated (26, 27). Type III collagen is abundant in the early stages of fibrosis, and upregulation of type I collagen is associated with later stages of fibrosis. Fibrosis in the liver results in collagen deposition and the release of propeptides, especially PIIINP. As a result, PIIINP is one of the best studied markers for fiber formation (28, 29, 30). Over the years, several radioimmunoassays have been developed to quantify PIIINP, with up to 94% sensitivity for cirrhosis detection and up to 81% specificity (31,32); All previous methods of analysis do not have neo-epitope specificity. In addition, analytical methods currently commercially available for the quantification of PIIINP use polyclonal antibodies or monoclonal antibodies targeting internal sequences of procollagen or a propeptide, and specifically identify the formation and / or degradation of collagen type III (31, 32).

Thus, in order to distinguish between the formation and degradation of collagen type III, the present inventors have discovered that neo-epitopes produced only during the formation process (i.e., fragments that are made during the formation of collagen type III but not during collagen type III degradation) It is necessary to measure and detect it.

WO 2014/170312 discloses monoclonal antibodies specific for the C-terminal PIIINP neo-epitope contained in the terminal amino acid of the C-terminal amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4), where X may be Gly or Pro.

Brocks (31) is in a modified Bordj C- terminal PIIINP sequence I C * E QSCPTGG NYSP-COOH (SEQ ID NO: 1); for (C * = acetamidomethyl protected Cys Gln search is replaced by Glu (E)) Although this antibody has been disclosed as a polyclonal antibody, it is non-specific to the terminal amino acid C-terminal sequence ICQSCPTGGQNYSP-COOH (SEQ ID NO: 2) of the bovine PIIINP and does not recognize human PIIINP.

Bayer (33) disclosed a sandwich ELISA using a monoclonal antibody as a detector for the sequence H ₂ N-GSPGPPGICQSCPTGPQNYSP-COOH (SEQ ID NO: 3), but binding epitopes are not defined.

Applicants have found that specific sandwich immunoassays using a neo-epitope specific antibody to the C-terminal neo-epitope of PIIINP disclosed in WO 2014/170312 can be used to determine the efficacy of a drug targeting lysyl oxidase (LOX), particularly a LOX antagonist drug , ≪ / RTI > Enzymatic collagen crosslinking by LOX and processing of pro-collagen are very important for tissue maturation and stability. In patients with long-term fibrosis, collagen is highly cross-linked and fibrosis is less likely to resolve. LOXL2, a specific LOX, is a major driving factor in pathophysiological collagen crosslinking in fibrotic tissue, and new LOXL2 antagonists are currently in clinical trials. Therefore, the analysis that can be used to evaluate the efficacy of drugs targeting LOX, such as LOX antagonists, will, of course, be a useful tool in the pharmaceutical industry.

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The present invention relates to a sandwich immunoassay method for detecting PIIINP cross-linked in a biological sample, wherein the cross-linked PIIINP comprises two or more PIIINP strands bound by cross-linking strands. The method comprises contacting a biological sample comprising a crosslinked PIIINP with a first monoclonal antibody bound to a surface, and adding a second monoclonal antibody, wherein the angle of the PIIINP contained in the crosslinked PIIINP Strand has a C-terminal neo-epitope of PIIINP formed by cleavage of intact < RTI ID = 0.0 > type III < / RTI > Two of the monoclonal antibodies specifically react to the C-terminal neo-epitope of PIIINP and the neo-epitope is included in the C-terminal amino acid sequence CPTGXQNYSP-COOH, where X is Gly or Pro. The method further comprises measuring a binding amount of the second monoclonal antibody.

The present invention also relates to a method for evaluating the efficacy of antagonist drugs targeting lysyl oxidase (LOX). The method comprises quantifying the amount of PIIINP cross-linked in two or more biological samples obtained from an individual at a first time point and at one or more subsequent time points during the administration of the antagonist drug to the subject using the sandwich immunoassay method described herein . The quantitative reduction of PIIINP cross-linked from the first time point to one or more subsequent time points during the administration of the antagonist drug means that it is an effective antagonist drug targeting LOX.

The present invention relates to kits for use in the sandwich immunoassay methods described herein. The kit comprises a first monoclonal antibody as described above and a solid support to which the labeled second monoclonal antibody described herein is conjugated.

Figure 1: Alignment of the sequence of the targeted PIIINP? 1 sequence in human (SEQ ID NO: 14) and rat (SEQ ID NO: 15) species (highlighted in box). Positions of the corresponding human (-) and rat (~) sequences in the? 1 chain of the N-terminal propeptide of type III collagen. Alignment was performed using NLP CLUSTALW software.
Figure 2: a) rat, which is recognized by the monoclonal antibody NB61N62 (lanes 1 and 3) and NB61N62 + selective peptide (lane 2 + 4), and b) a specific example of an N-terminal propeptide of type III collagen in a human- Western blot showing bands. Two bands of about 52-60 kDa were observed in the rats, whereas one band was observed in humans. The addition of selective peptides decreased band strength in both rats and humans.
Figures 3A-3D are results of a PRO-C3 ELISA demonstrating a typical calibration curve and native reactivity for human, rodent and mouse material. Figure 3A: Inhibition of competitive PRO-C3 ELISA using calibration curve and healthy human serum, plasma and amniotic fluid (AF). The calibrator curve was diluted 2 fold from 76.31 ng / mL while the natural material was diluted from 1: 2 to 1:16 as indicated by (-). Figure 3B: Inhibition of Competitive PRO-C3 ELISA using calibration curve and healthy rat serum, plasma and AF. The calibrator curves were diluted 2-fold from 200 ng / mL, while the natural material proceeded 1: 8 in non-dilution as indicated by (-). Figure 3C: Inhibition of Competitive PRO-C3 ELISA using calibration curve and healthy mouse serum and plasma. The calibrator curve was diluted 2-fold at 200 ng / mL, while the native material was diluted 1: 4 in non-dilution as indicated by (-). Figure 3D: Neo-epitope specificity of PIIINP neo-epitope specific antibodies using peptide sequences of extended peptides, i.e., calibration peptides with one amino acid at the C-terminal end. Calibration curves, extended peptides and nonsense peptides were diluted 2-fold at 76.31 ng / mL. The signal is observed with an optical density at 450 nm limiting the 650 nm background as a function of the peptide concentration.
Figure 4: Results of an in vitro model of lung fibroblasts ("scar-in-a-jar").
Figure 5: Comparison of Pro-C3X levels in extracts of keloid and extracts of normal skin.
6A-6B: Experimental results on patients with liver fibrosis.
Figure 7: Diagram showing the Pro-C3X analysis.
Figure 8: Experimental results for patients with alcoholic fatty liver disease.
Figure 9: Pro-C3X levels of supernatant collected on day 10 in a Scar-in-a-Jar model. Significance was assessed by one-way ANOVA using Dunnett's multiple comparison test comparing each condition with TGF-beta alone. Data are expressed as mean and SD. **** p <0.0001. BAPN,? -Aminopropionitrile; TGF-β, Transforming Growth Factor β.

As used herein, the term "neo-epitope" refers to an N- or C-terminal peptide located at the end of the polypeptide, i.e., located at the N- or C-terminus of the polypeptide, It is not.

As used herein, the term "competitive ELISA " refers to competitive enzyme-linked immunosorbent assays and techniques known to those skilled in the art.

As used herein, the term " sandwich immunoassay method "refers to the use of two or more antibodies to detect an antigen in a sample and is a technique known to those skilled in the art.

The term monoclonal antibody NB61N-62 herein refers to the neo-epitope specific antibody to the C-terminal neo-epitope of PIIINP and the neo-epitope refers to the C-terminal sequence CPTGXQNYSP-COOH where X is Gly or Pro (SEQ ID NO: 4).

As used herein, the term "PRO-C3" is used to distinguish the PIIINP assay described herein from PIIINP assays known in the art that are not based on the specific binding of neo-epitopes originating from PIIINP.

As used herein, the term "PRO-C3X" assay refers to the sandwich immunoassay described herein for detecting and quantifying cross-linked PIIINP.

Monoclonal antibodies suitable for use in the methods of the invention are described in WO 2014/170312, which specifically reacts with the C-terminal neo-epitope of PIIINP and the neo-epitope comprises a C-terminal with X being Gly or Pro Is included in the amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) and the monoclonal antibody does not substantially recognize or bind an extended version of the C-terminal amino acid sequence, CPTGXQNYSPQZ-COOH (SEQ ID NO: 5) Or at least one amino acid of collagen type III sequence.

Preferably, the monoclonal antibody is a neo-epitope of human PIIINP produced from intact procollagen type III by cleavage of PIIINP at the Pro-Gln binding site between amino acids P153-Q154 of human PIIINP by N-protease And specifically reacts with the C-terminal sequence CPTGPQNYSP-COOH (SEQ ID NO: 6).

In another embodiment, the monoclonal antibody is a Neo-epitope C of rodent PIIINP, which is formed from intact procollagen type III by N-protease cleavage of PIIINP at the Pro-Gln binding site between amino acids P154-Q155 of rodent PIIINP. -Terminal sequence CPTGGQNYSP-COOH (SEQ ID NO: 7).

Preferably the ratio of affinity of the monoclonal antibody to the amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) for the affinity of the monoclonal antibody for the extended amino acid sequence CPTGXQNYSPQZ-COOH (SEQ ID NO: 5) is at least 10 to 1, Preferably at least 100 to 1, more preferably at least 1,000 to 1, more preferably at least 10,000 to 1, more preferably at least 100,000 to 1, most preferably at least 1,000,000 to 1.

Preferably, the monoclonal antibody does not recognize or bind a shortened version of the C-terminal neo-epitope of PIIINP with the amino acid sequence CPTGXQNYS (SEQ ID NO: 8).

Preferably the ratio of affinity of the monoclonal antibody to the amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) for the affinity of the monoclonal antibody for the reduced amino acid sequence CPTGXQNYS-COOH (SEQ ID NO: 8) is at least 10 to 1, Preferably at least 100 to 1, more preferably at least 1,000 to 1, more preferably at least 10,000 to 1, more preferably at least 100,000 to 1, most preferably at least 1,000,000 to 1.

The present invention relates to a sandwich immunoassay method for detecting in a biological sample a crosslinked PIIINP comprising two or more strands of PIIINP bound by inter-strand crosslinking,

Contacting a biological sample comprising a cross-linked PIIINP with a first monoclonal antibody bound to a support, wherein each strand of PIIINP contained in the cross-linked PIIINP is produced by N-protease cleavage of intact type III procollagen A C-terminal neo-epitope of PIIINP;

Adding a second monoclonal antibody; And

And measuring the binding amount of the second monoclonal antibody,

Both the first monoclonal antibody and the second monoclonal antibody specifically react to the C-terminal neo-epitope of PIIINP, and the neo-epitope is included in the C-terminal amino acid sequence CPTGXQNYSP-COOH where X is Gly or Pro.

Preferably, the monoclonal antibody does not exhibit substantial recognition or binding to an extended version of the C-terminal amino acid sequence, i. E. CPTGXQNYSPQZ-COOH, wherein Z is omitted or one or more of the amino acids of the collagen type III sequence.

The sandwich immunoassay methods described herein use the same antibody as both the capture antibody and the detection antibody, and thus double-stranded peptides (i.e., cross-linked) can be identified by analysis.

Preferably, the amount of PIIINP crosslinked in the biological fluid is quantified using a sandwich immunoassay, and the biological fluid may be serum, plasma, urine, amniotic fluid, tissue supernatant, or cell supernatant, but is not limited thereto.

The sandwich immunoassay may be, but is not limited to, radioimmunoassay, fluorescent immunoassay, or enzyme-linked monoclonal antibody.

In a preferred embodiment, the second monoclonal antibody may be labeled to determine the binding amount of the second monoclonal antibody.

Preferably, the second monoclonal antibody may be an enzyme-linked antibody. The enzyme may be, but is not limited to, horseradish peroxidase (HRP).

Preferably, the second monoclonal antibody is radiolabeled or a fluorophore is attached.

While these materials are preferred labeling materials for use with the present invention, it is contemplated that any suitable labeling system may be used, such as a non-exclusive example, such as a DNA reporter or electrochemiluminescent tag.

In other embodiments, the amount of binding of the second monoclonal antibody can be measured using an additional labeled antibody that recognizes the second monoclonal antibody. Additional labeled antibodies may be labeled with a labeling substance as described above.

In a preferred embodiment of the invention, the sandwich immunoassay method further comprises the step of associating the amount of cross-linked PIIINP as measured by the present method with a standard sample of fibrotic disease for which the severity of illness is known to assess the severity of the fibrotic disease . Such fibrotic disease may be, but is not limited to, liver disease.

In another aspect, the sandwich immunoassay method described herein can be used in a method for evaluating the efficacy of drugs targeting lysyl oxidase (LOX), such as antagonists targeting LOX.

Accordingly, the present invention also relates to a method for evaluating the efficacy of an antagonist drug targeting lysyl oxidase (LOX), the method comprising administering to the subject a therapeutically effective amount of a PIIINP Wherein the biological sample is obtained from an individual at a first time point and at a subsequent time point thereafter, during a period of administration of the antagonist drug to the subject, wherein the biological sample is administered from the first time point onward A quantitative decrease in cross-linked PIIINP to one or more subsequent time points means that the antagonist drug is an effective antagonist drug targeting LOX.

Preferably, the method quantifies the efficacy of the antagonist drug.

Preferably, the method evaluates the efficacy of an antagonist drug targeting LOXL2.

In another aspect, the present invention relates to a kit for use in the sandwich immunoassay method described herein, which comprises a solid support to which the first monoclonal antibody as described above is bound and a second monoclonal antibody as described above.

Example

Materials and General Considerations

All reagents used in the experiments are high-quality standard chemicals purchased from companies such as Merck (Whitehouse Station, NJ, USA) and Sigma Aldrich (St. Louis, Mo., USA). The synthetic peptides used for monoclonal antibody production and validation are as follows: 1) immunogenic peptides: Ovalbumin (OVA) -CGG-CPTGPQNYSP (SEQ ID NO: 10), 2) Screening peptides: Biotin-CGG-CPTGPQNYSP 11), and 3) the selected peptide: CPTGPQNYSP (SEQ ID NO: 6). Synthetic peptides were purchased from Chinese Peptide Company in Beijing, China.

Example  1 - monoclonal antibody NB61-N62

Monoclonal antibody production

The sequence of the N-terminal propeptide of type III collagen was aligned between human, rat and mouse species and was selected as species homology by protein blasting and uniqueness in other ECM proteins. The amino acid sequence 145'-CPTGPQNYSP-'153 (SEQ ID NO: 6) in the α1 chain PIIINP has 100% human and rat homology (FIG. Monoclonal antibodies were prepared by infecting Balb / C mice at 4-5 weeks of age with 200 쨉 l of the emulsified antigen and 50 쨉 g of the PIIINP neo-epitope C-terminal sequence (OVA-CGG-CPTGPQNYSP (SEQ ID NO: 10) Using a reinforcement body. Immunization was performed every two weeks until a stable serum titer level was reached. Mice displaying the highest serum titer were selected for fusion. Mice were rested for one month and then intravenously boosted with 50 [mu] g of the PIIINP neo-epitope C-terminal sequence in 100 [mu] l of 0.9% sodium chloride solution 3 days before isolating the spleen. Hybridomas were prepared as described in (34) by fusing spleen cells with SP2 / 0 myeloma cells and cloned in culture dishes using the semi-medium method. Clones were inoculated into 96-well microtiter plates for further propagation by applying a limiting dilution method to multiply a single clone. The reactivity to calibrator peptides and natural substances in the supernatant was screened by indirect ELISA using streptavidin-coated plates. Biotin-CGG-CPTGPQNYSP (SEQ ID NO: 11) was used as a screening peptide and the free peptide CPTGPQNYSP (SEQ ID NO: 6) was used as a calibrator for additional specificity assays for clones.

Characterization of clones

In a preliminary ELISA using 2 ng / ml biotinylated peptide and a supernatant of monoclonal hybridoma cells under proliferation in a streptavidin-coated microtiter plate, the native reactivity and affinity of the peptide , Serum and amniotic fluid (AF), were analyzed in humans and rats. Human AF was obtained from 30 women who underwent selective cesarean section of the lower uterus in Beijing Obstetrics Hospital for 2 months. AF 100-200 ml were collected directly after incision, and the amniotic fluid was stored at -20 ° C until use. Experiments were approved by the local ethics committee, and all women asked for written consent before sampling. Rat AF was drained from the womb of a pregnant Wistar rat two days before the scheduled date. Antibody specificity was checked by preliminary analysis with deselection peptide and extended peptide (i. E. Calibrator peptides each with 10 amino acid substitutions and calibrator peptides with one amino acid added to the cuts). Isotype of monoclonal antibody was determined by Clonotyping System-HRP kit cat. 5300-05 (Southern Biotech, Birmingham, AL, USA).

Characterization of antibodies

Before Western blotting, the total protein concentration of human and rat AF was measured using BCA (Bicinchoninic acid) protein analysis according to the manufacturer's instructions. Briefly, BCA was diluted 2-fold from 2 mg / ml in PBS to create standard columns for sample calibration. Samples were diluted 1: 4 in 1x phosphate-buffered saline (PBS) and 25 [mu] l of the sample was added to the microtiter plate with 200 [mu] l of working reagent (reagent A and B mixed at 50: 1). The contents were mixed on a plate stirrer for 30 seconds and then incubated at 37 DEG C for 30 minutes. After the incubation, the plate was cooled to room temperature and the absorbance was measured at 562 nm (Molecular Devices, SpectraMax M, CA, USA) in an ELISA reader. Thereafter, rat or human AF was mixed with sample buffer (x2) and reducing agent (x10), heated at 70 DEG C for 10 minutes and then subjected to 4-20% tris-glycine sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS -page) and was run at 180 V for 1 hour. Protein bands were blotted onto a nitrocellulose membrane using the Invitrogen i-Blot gel transfer system according to the manufacturer's instructions. The membranes were blocked in blocking buffer (5% skim milk / Tris-buffered saline + Tween (TBST) overnight at 4 ° C and incubated with 1 μg / ml horseradish peroxidase (HRP) -modified PIIINP neo- And incubated with the epitope-specific monoclonal antibody NB61N-62. Excess PIIINP neo-epitope calibrator peptides and antibodies were added at a ratio of 10: 1 to investigate the specificity of the PIIINP neo-epitope specific monoclonal antibody, preincubated for 1 hour, added to the membrane and incubated overnight. After incubation, the membranes were washed with TBST for 4 x 10 minutes, incubated with 4 ml chemiluminescence detection kit (ECL) and developed using Amersham Hyperfilm.

Clone selection and characterization

Subtypes were identified as IgGl subtypes. Western blot analysis showed that the PIIINP neo-epitope specific monoclonal antibody NB61N-62 detected two bands of about 52-60 kDa molecular size in the amniotic fluid of the rat while only one band of about 52 kDa was detected in human amniotic fluid . In addition, signals were partially inhibited by selective peptides in rats and could be inhibited in humans (Fig. 2). Native reactivity was observed using an NB61N-62 antibody in an ELISA. Native reactivity to rodent, plasma and AF as well as native reactivity to human serum, plasma and AF were observed (Figures 3A-3C). Signals were somewhat reduced in mouse serum and plasma. Competitive ELISA signals were inhibited from 1: 2 to 1:16, 1: 8 in non-diluted, or 1: 4 in non-diluted, respectively, in human, rodent and mouse native materials. In all three species after dilution of the natural material, the same dilution pattern as the killer curve was obtained. Human AF inhibited signals up to 100%; 80% for Rat AF; 70% for human serum and plasma and rat serum; 44% in rat plasma and 35% in mouse serum and plasma. 0 inhibition was observed in the extended peptide (CPTGPQNYSPQ (SEQ ID NO: 6)) and nonsense peptide (GSPGKDGVRG (SEQ ID NO: 12)) (FIG. 3D).

Example  2 - NB61N PRO-C3 ELISA using -62

The supernatant was collected from the hybridoma producing antibody and monoclonal antibodies were purified using a HiTrap affinity column (GE Healthcare Life Science, Little Chalfont, Buckinghamshire, UK) and then purified using Lightning-Link HRP conjugation kit (Innova Biosciences , Babraham, Cambridge, UK) were labeled with HRP using the manufacturer's instructions.

The PRO-C3 competitive ELISA procedure is as follows: Roche's cat. 11940279 96-well streptavidin-coated ELISA plates were coated with biotinylated peptides dissolved in cotter buffer (50 mM PBS-BTE + 10% sorbitol, pH 7.4) Biotin-CGG-CPTGPQNYSP (SEQ ID NO: 11), incubated at 20 ° C for 30 minutes under dark conditions, and then rinsed with washing buffer (20 mM Tris, 50 mM NaCl, pH 7.2). Then 20 μl of the peptide calibrator or sample was added to the appropriate well and 100 μl of the HRP-conjugated monoclonal antibody NB61N-62 dissolved in incubation buffer (50 mM PBS-BTB + 10% LiquidII (Roche), pH 7.4) Was added and the plate was incubated at 4 [deg.] C for 20 hours and then washed. Finally, 100 μl of tetramethylbenzidine (TMB) (Kem-En-Tec cat .: 438OH) was added and the plate was incubated for 15 minutes at 20 ° C under dark conditions and the stop solution (1 % H ₂ SO ₄ ) was added and the plate was analyzed at 450 nm in an ELISA reader and analyzed at 650 nm as a reference (Molecular Devices, SpectraMax M, CA, USA). The calibration curve was created using a 4-parametric mathematical fit model.

Technical evaluation

Linearity was determined using twofold dilutions of healthy serum and plasma samples and calculated as the percent recovery of 100% sample. Antibody specificity was calculated as the recovery of 100% calibrator peptide (CPTGPQNYSP (SEQ ID NO: 6)), extended peptide (CPTGPQNYSPQ (SEQ ID NO: 13)) and nonsense peptide (GSPGKDGVRG (SEQ ID NO: 12)). The lower detection limit (LLOD) was calculated from the mean of blank + 3x standard deviation (SD) from the 21 measurements of standard K. The detection upper limit (ULOD) was determined as the average of 10 measurements of standard A - 3xSD. The lower limit of quantification (LLOQ) was obtained as the lowest measured concentration repeatedly less than 30% accuracy. The intra-assay and inter-assay deviations were measured through 10 independent measurements of 8 QC samples, each of which consisted of a double measurement of the sample. The accuracy of the samples was measured in healthy human serum samples with or without significant concentrations of human amniotic fluid and calculated as the percentage of the theoretical amount of serum to be recovered. Interference was measured in healthy human serum supplemented with significant concentrations of hemoglobin, lipemia and biotin and calculated as percent theoretical amount of serum.

result

The measurement range of the human PRO-C3 ELISA was determined by calculating ULOD and LLOQ to provide a range of 0.867-60.1 ng / ml. The technical performance of the PRO-C3 ELISA showed acceptable tolerance of analytical and analytical variances of 11.03% and 4.11% (Table 1), with acceptable ranges of 15% and less than 10%, respectively.

Table 1: Inter-assay and intra-assay variance in PRO-C3 assay using human serum quality control sample # 1-8 (HS1-HS8). The deviation was calculated as the mean deviation between each of the 10 measurements for each sample.

Sample value
(ng / mL) Variance within analysis% Variance between analyzes% HS1 24.24 2.28 5.94 HS2 11.62 2.90 6.45 HS3 8.40 5.31 11.99 HS4 6.54 4.46 11.31 HS5 6.36 3.88 13.09 HS6 5.23 3.98 12.31 HS7 4.29 3.53 12.94 HS8 2.98 4.66 18.56

Dilution recovery was performed using healthy serum and plasma samples from human, rat and mouse. The number of dilutions was within the acceptable 100 ± 20% recovery rate (Table 2). Additional dilutions yielded measurements below LLQ.

Table 2: Dilution recovery of PRO-C3 assay using human, rat and mouse samples. Human serum (HS), human plasma (HP), rat serum (RS), mouse serum (MS), mouse plasma (MP).

PIIINP
ng / ml HS
(n = 2) HP
(n = 3) RS
(n = 10) MS
(n = 2) MP
(n = 2) Non-diluted 100% - 100% 100% - Dilution 1: 2 98 100% 116 96 100% Dilution 1: 4 103 91 110 118 114 Dilution 1: 8 114 87 - - - Dilution 1:16 - 92 - - - Average 105 90 113 107 114

As a result of calibrator peptide addition to serum or plasma, the average recoveries were 56% and 55%, respectively (Table 3).

Table 3: Calibrator peptides in human serum or plasma, and spiking recovery of human AF in human serum or plasma. Recovery was calculated as percent recovery of calculated peptide / AF in pure serum / plasma versus serum / plasma. The concentrations of the calibrator peptides were 38.16 ng / ml (StdB), 19.08 ng / ml (StdC), 9.54 ng / ml (StdD), 4.77 ng / ml (StdE), 2.39 ng / StdG). AF was started at 1: 2 and added in two-fold dilutions.

Added standard Serum (n = 3)
sRE% Plasma (n = 3)
sRE% AF added Serum (n = 3)
sRE% Plasma (n = 3)
sRE% StdB 16 15 2x 101 103 StdC 29 25 4x 103 108 StdD 42 38 8x 106 113 StdE 58 54 16x 103 112 StdF 70 69 32x 104 115 StdG 82 83 64x 103 110 Buffer 92 100 Buffer 99 104 Average sRE% 56 100 Average sRE% 55 111

However, the average recovery was 100% and 111%, respectively, as a result of human AF addition with two-fold dilutions starting at 1: 2 in healthy human serum or plasma. No interference was observed in serum with various concentrations of hemoglobin, biotin and serum lipids (Table 4).

Table 4: Interference of hemoglobin, serum lipids and biotin in human serum added at various concentrations. All data are expressed as% pure serum versus recovery.

hemoglobin
mmol / L RE% Serum lipid
mmol / L RE% Biotin
ng / L RE% 0.5 68 0.56 101 160,000 134 0.25 74 0.28 103 80,000 113 0.13 81 0.14 99 40,000 96 0.063 81 0.07 104 20,000 97 0.031 82 0.04 101 10,000 94 0.016 86 0.00 100 5,000 87 0.008 95 2,500 100 0.000 100 0 100 Average 83 101 103

The stability of the analyte was acceptable for up to four cycles of freezing / thawing, and the recovery was 100 ± 20% compared to one freezing / thawing cycle (Table 5).

Table 5: Stability of three human serum and plasma samples in four freeze / thaw cycles. All data are expressed as% of the average number of times of one freeze / thaw cycle.

Frozen / thawed cycle serum
Average Recovery Rate% EDTA plasma
Average Recovery Rate% Heparin plasma
Average Recovery Rate% Site-rate plasma
Average Recovery Rate% One 100% 100% 100% 100% 2 103 102 103 109 3 99 99 98 103 4 102 100 98 100

Example  Determination of the ratio of 3-binding affinity

Binding Affinity of Monoclonal Antibodies to Target Sequence: To obtain the ratio of binding affinities of monoclonal antibodies to extended or reduced sequences, each sequence was synthesized and analyzed by PRO-C3 ELISA As a calibrator peptide. The IC ₅₀ values of each sequence / antibody combination are determined using the calibration curves obtained. The ratio of IC ₅₀ [target] / IC ₅₀ [extended or reduced] defines the ratio of binding affinities.

Example  4 - PRO- C3X  analysis

As described above, enzymatic collagen crosslinking by lysyl oxidase (LOX) and processing of procollagen are important for tissue maturation and stability. Thus, strand cross-linking monitoring of procollagen type III prior to enzymatic processing can prove useful in monitoring in vivo activity of LOX. This can be achieved by detecting and quantifying cross-linked PIIINP (i. E., Above the PIIINP strand bound by interchain linkage formed by LOX in procollagen type III before enzymatic processing of procollagen). A high level of cross-linked PIIINP detected in the circulation will mean that the LOX activity is higher. That is, monitoring the level of cross-linked PIIINP in a drug experiment for a drug targeting LOX, for example, a LOX antagonist drug, can provide drug efficacy data.

ELISA

The streptavidin-coated plate was coated with 100 μl / well of 1 μg / ml biotinylated capture antibody (biotin-linked NB61-N62) and incubated at 20 ° C. with stirring at 300 rpm for 30 minutes. The plates were rinsed five times with wash buffer (20 nM TRIS, 50 mM NaCl, pH 7.2). After adding the sample standard material or the control (20 ㎕), immediately add 100 ㎕ of assay buffer and incubate at 4 캜 with stirring at 300 rpm for 20 hours. After incubation, the plates were rinsed five times with wash buffer. 1 μg / ml HRP-labeled detection antibody (HRP-linked NB61-N62) was added at 100 μl / well and incubated at 20 ° C. with stirring for 1 hour at 300 rpm. After incubation, the plates were rinsed five times with wash buffer. 100 ㎕ of 3,3 ', 5,5'-tetramethylbenzidine (TMB) was added and incubated at 20 캜 for 15 minutes under dark conditions. To stop the enzymatic reaction of TMB, 100 [mu] l 0.1% sulfuric acid was added. The enzyme reaction was then read in an ELISA reader using a quadratic curve fit. Each ELISA plate included both the kit control and in-house quality control samples, and the inter-assay deviation was monitored. All samples were measured within the scope of specific analysis. All samples below the lower limit of quantification (LLOQ) were assigned the LLOQ value.

The technical characteristics of the analysis are as follows:

parameter result Biological substrate Serum, plasma, supernatant, extract Deviation within analysis 2% (acceptable for <10%) Deviation between analyzes 6% (acceptable for <15%) Measuring range 0.965-17.586 ng / ml Lower limit of detection 0.251 ng / ml Normal range in healthy serum 4.022 (+ - 2.24) ng / ml Required volume 30 [mu] l Serum / Plasma; 60 [mu] l supernatant / extract Spiking recovery Serum peptides: 94%
Serum in serum: 86%

result

Scar tissue

Preliminary results ("scar-in-a-jar") using an in vitro model of lung fibroblasts suggest strongly that LOX family enzymes are responsible for cross-linking in PIIINP (TGF- ) &Lt; / RTI &gt; enzymatic activity). Briefly, Pro-C3X (i.e., cross-linked PIIINP) was produced by culturing 5 days of pulmonary fibroblasts under a crowded condition and TGF-beta stimulation. Pro-C3X was significantly increased after 12 days of incubation with TGF-β, and Pro-C3X was little observed in the absence of TGF-β (FIG. 4). Similarly, Pro-C3X was increased in the keloid extract compared to normal skin extract (Fig. 5).

Liver fibrosis

Experiments on patients with liver fibrosis were performed using the "Pro-C3X" assay and compared with the "Pro-C3" competitive ELISA described herein. Pro-C3X was found to be significantly increased in the later stages of the disease, a more severe condition of fibrosis, similar to that of healthy controls at early stages (Fig. 6A). In contrast, the level of Pro-C3 was different at all stages of the disease (Fig. 6B).

The selectivity difference between Pro-C3X and Pro-C3 analyzes is based on the fact that the Pro-C3X assay recognizes only the cross-linked PIIINP whereas the Pro-C3 assay recognizes both cross-linked and non-cross-linked PIIINP. Figure 7 shows Pro-C3X, pictorially illustrating the basis of this conclusion:

Pro-C3X analysis: In the presence of cross-linked PIIINP, the first antibody will bind to the free epitope on the first strand of PIIINP, and then the second antibody binds to the glass epitope of the second strand of PIIINP . However, in the presence of the non-crosslinked PIIINP, the surface-bound antibody binds to the non-crosslinked collagen type III free epitope, but the second antibody fails to bind because the binding epitope is already occupied, Therefore, when the second antibody is added, signal generation fails. Thus, the signal of the Pro-C3X assay is only due to the detection of cross-linked PIIINP.

Conversely, in the Pro-C3 assay, the antibody will bind to the PIIINP strand substantially all of which includes the binding free epitope, regardless of whether PIIINP is cross-linked or not. Thus, the signal obtained from the Pro-C3 analysis is an aggregate signal of cross-linked PIIINP and non-cross-linked PIIINP.

That is, this selectivity prompts the use of the Pro-C3X assay in evaluating the efficacy of drugs targeting LOX; Monitoring of the PIIINP cross-linking level, which appears to be the result of LOX activity during drug administration to an individual, can be used to monitor drug activity to monitor drug efficacy.

Alcoholic Fatty hepatitis

Experiments on patients with alcoholic steatohepatitis were performed using the "Pro-C3X" assay and compared with the "Pro-C3" assay described herein. In Alcoholic Fatty Hepatitis, both Pro-C3 and Pro-C3X were increased in the late stages of disease (Metavir 2-4). However, Pro-C3 was not correlated with the MELD score (P = 0.527) in patients with cirrhosis, but Pro-C3X had a strong correlation (correlation coefficient 3.34, P <0.001). ProC3 has a negative correlation with monoalbumin, but ProC3X has a correlation with albumin, bilirubin, and gamma-glutamyl transpeptidase (GGT) (Fig. 8). Pro-C3X increase in later stages suggests an increase in PIIINP bridging, which will be consistent with increased liver scarring (ie, increased LOX activity).

Example  5 In the Scar-in-a-Jar model, the Pro- C3X  evaluation

Background : Fibrosis is the accumulation of extracellular matrix (ECM) in the affected tissue, which can lead to organ failure and ultimately death. For example, after stimulation with transforming growth factor (TGF) -ß, fibroblasts are the main cell type causing excessive accumulation of ECM proteins, particularly collagen. Here, we have found that the use of the in vitro model "Scar-in-a-Jar" known to form ECMs and bridges in combination with the Pro-C3X ELISA to investigate collagen formation and crosslinking formation during fiber formation Lt; / RTI &gt; The tool can be used to investigate new anti-fibrous compounds by analyzing the regulation of the fibrous process.

METHODS : Healthy human lung fibroblasts (L248) were inoculated at confluence with 30,000 cells / well of cells. Cells were cultured for 18 days in DMEM containing 0.4% FCS, 225 mg / mL ficoll 70, 150 mg / mL ficoll 400 and 1% ascorbic acid. Cells were stimulated with 1 ng / mL TGF-beta alone or with lysyloxydase (LOX) inhibitor beta -aminopropionitrile (BAPN; 0.02 or 0.2 mM) to inhibit cross-linking formation. Non-stimulated cells or cells cultured in ficoll non-containing media were used as controls. The medium was replaced on days 3, 6, 10 and 14. Fibroblast survival was assessed by AlamarBlue analysis. Pro-C3X levels were analyzed in supernatants collected using the Pro-C3X sandwich ELISA described above.

Results : TGF-β stimulation induced the release of Pro-C3X from day 3, and the level of Pro-C3X reached its peak at 10 days, 14-fold higher than that of non-stimulated cells (p <0.0001; 9). 0.02 mM BAPN treatment had no significant effect and 0.2 mM BAPN induced a significant decrease in the level of Pro-C3X (0.59-fold change, p <0.0001; Fig. 9) compared with TGF-beta stimulation alone.

CONCLUSION : The pan LOX inhibitor BAPN significantly reduced the level of Pro-C3X at a concentration of 0.2 mM, which means that the Pro-C3X ELISA analyzes the cross-linked epitope. Thus, Pro-C3X ELISA can be used to assess fibroblast activity and thus screen for potential anti-fibrotic compounds. TGF-beta stimulation induced the release of Pro-C3X, a marker of cross-linked collagen type III pro-peptide.

In conclusion, the Pro-C3X assay is a second generation assay that combines the cleavage neo-epitope of collagen type III pro-peptide with the presence of intramolecular crosslinking. This analysis describes the crosslinking of collagen molecules in other processes, ie scars, in the fiber formation timeline, thus providing additional information on the Pro-C3 measurement. Thus, as the use of LOX inhibitors has been shown to reduce the presence of cross-linked PIIINP biomarkers, Pro-C3X analysis can be used to examine the efficacy of drugs targeting LOX, particularly LOX antagonist / inhibitor drugs.

In this specification, the term &quot; or &quot;, as used herein, refers to the case where the operator &quot; exclusively or &quot; meets either one or both of the conditions mentioned, Is used as an operator. The term 'comprising' is used to mean 'including' rather than 'consisting of'. The contents of all the above-mentioned prior art are incorporated herein by reference. The content of any existing published literature here should not be construed as an admission or reference to the teaching that is at this time a general knowledge common in Australia or elsewhere.

               SEQUENCE LISTING <110> Nordic Bioscience A / S <120> PIIINP Neo-epitope Assay <130> P19876WO &Lt; 150 > US15 / 014,241 <151> 2016-02-03 <160> 15 <170> BiSSAP 1.3.6 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Prior art peptide <220> <221> SITE <222> 2 <223> acetamido protected Cys <400> 1 Ile Cys Gln Ser Cys Pro Thr Gly Gly Glu Asn Tyr Ser Pro 1 5 10 <210> 2 <211> 14 <212> PRT <213> Bovine <220> <223> Bovine PIIINP C-terminal sequence <400> 2 Ile Cys Gln Ser Cys Pro Thr Gly Gly Gln Asn Tyr Ser Pro 1 5 10 <210> 3 <211> 21 <212> PRT <213> Homo sapiens <220> <223> C-terminal PIIINP sequence <400> 3 Gly Ser Pro Gly Pro Pro Gly Ile Cys Gln Ser Cys Pro Thr Gly Pro 1 5 10 15 Gln Asn Tyr Ser Pro             20 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Antibody epitope <220> <221> SITE <222> 5 <223> Xaa can be Pro or Gly <400> 4 Cys Pro Thr Gly Xaa Gln Asn Tyr Ser Pro 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Elongated epitope peptide <220> <221> SITE <222> 5 <223> Xaa can be Pro or Gly <220> <221> SITE <222> 12 <223> Xaa can be absent or can be one or more amino acids of the       sequence of collagen type III <400> 5 Cys Pro Thr Gly Xaa Gln Asn Tyr Ser Pro Gln Xaa 1 5 10 <210> 6 <211> 10 <212> PRT <213> Homo sapiens <220> <223> Human PIIINP neo-epitope C-terminal sequence <400> 6 Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro 1 5 10 <210> 7 <211> 10 <212> PRT <213> Rodentia sp. <220> <223> Rodent PIIINP neo-epitope C-terminal sequence <400> 7 Cys Pro Thr Gly Gly Gln Asn Tyr Ser Pro 1 5 10 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Shortened epitope peptide <220> <221> SITE <222> 5 <223> Xaa can be Pro or Gly <400> 8 Cys Pro Thr Gly Xaa Gln Asn Tyr Ser 1 5 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> 1 <223> Xaa is absent or is biotinylated-Cys Gly Gly <220> <223> Biotinylated peptide <220> <221> SITE <222> 2 <223> Cys is biotinylated if Xaa is absent <400> 9 Xaa Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro 1 5 10 <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> 1 <223> Cys has N-terminal bound ovalbumin <220> <223> Ovalbumin bound <400> 10 Cys Gly Gly Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro 1 5 10 <210> 11 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> 1 <223> Cys is N-terminal biotinylated <220> <223> Biotinylated peptide <400> 11 Cys Gly Gly Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Non-sense peptide <400> 12 Gly Ser Pro Gly Lys Asp Gly Val Arg Gly 1 5 10 <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> elongated peptide <400> 13 Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro Gln 1 5 10 <210> 14 <211> 179 <212> PRT <213> Homo sapiens <400> 14 Met Met Ser Phe Val Gln Lys Gly Ser Trp Leu Leu Leu Ala Leu Leu 1 5 10 15 His Pro Thr Ile Ile Leu Ala Gln Gln Glu Ala Val Glu Gly Gly Cys             20 25 30 Ser His Leu Gly Gln Ser Tyr Ala Asp Arg Asp Val Trp Lys Pro Glu         35 40 45 Pro Cys Gln Ile Cys Val Cys Asp Ser Gly Ser Val Leu Cys Asp Asp     50 55 60 Ile Ile Cys Asp Asp Gln Glu Leu Asp Cys Pro Asn Pro Glu Ile Pro 65 70 75 80 Phe Gly Glu Cys Cys Ala Val Cys Pro Gln Pro Pro Thr Ala Pro Thr                 85 90 95 Arg Pro Pro Asn Gly Gln Gly Pro Gln Gly Pro Lys Gly Asp Pro Gly             100 105 110 Pro Pro Gly Ile Pro Gly Arg Asn Gly Asp Pro Gly Ile Pro Gly Gln         115 120 125 Pro Gly Ser Pro Gly Ser Pro Gly Pro Pro Gly Ile Cys Glu Ser Cys     130 135 140 Pro Thr Gly Pro Gln Asn Tyr Ser Pro Gln Tyr Asp Ser Tyr Asp Val 145 150 155 160 Lys Ser Gly Val Ala Val Gly Gly Leu Ala Gly Tyr Pro Gly Pro Ala                 165 170 175 Gly Pro Pro              <210> 15 <211> 178 <212> PRT <213> Rattus <400> 15 Met Met Ser Phe Val Gln Cys Gly Thr Trp Phe Leu Leu Thr Leu Leu 1 5 10 15 His Pro Ser Leu Ile Leu Ala Gln Gln Ser Asn Val Asp Glu Leu Gly             20 25 30 Cys Asn Tyr Leu Gly Gln Ser Tyr Glu Ser Arg Asp Val Trp Lys Pro         35 40 45 Glu Pro Cys Gln Ile Cys Val Cys Asp Ser Gly Ser Val Leu Cys Asp     50 55 60 Asp Ile Met Cys Asp Asp Glu Pro Leu Asp Cys Pro Asn Pro Glu Ile 65 70 75 80 Pro Phe Gly Glu Cys Cys Ala Ile Cys Pro Gln Pro Ser Thr Pro Ala                 85 90 95 Pro Val Ile Pro Asp Gly Asn Arg Pro Gln Gly Pro Lys Gly Asp Pro             100 105 110 Gly Pro Pro Gly Ile Pro Gly Arg Asn Gly Asp Pro Gly Leu Pro Gly         115 120 125 Gln Pro Gly Leu Pro Gly Pro Pro Gly Ser Pro Gly Ile Cys Glu Ser     130 135 140 Cys Pro Thr Gly Gly Gln Asn Tyr Ser Pro Gln Phe Asp Ser Tyr Asp 145 150 155 160 Val Lys Ser Gly Val Gly Gly Met Gly Gly Tyr Pro Gly Pro Ala Gly                 165 170 175 Pro Pro         

Claims (16)

A sandwich immunoassay method for detecting cross-linked PIIINP (PIIINP) cross-linked in a biological sample,
Wherein said crosslinked PIIINP comprises two or more PIIINP strands bound by inter-strand cross-linking,
The method comprises:
Contacting a biological sample comprising the crosslinked PIIINP with a first monoclonal antibody bound to a surface;
Adding a second monoclonal antibody; And
And measuring the binding amount of the second monoclonal antibody,
Each of the strands of PIIINP contained in the crosslinked PIIINP is a C-terminal neo-epitope of PIIINP formed by cleavage of intact type III procollagen by N-protease. / RTI &gt;
Wherein said first monoclonal antibody and said second monoclonal antibody specifically react with a C-terminal neo-epitope of PIIINP,
Wherein the neo-epitope is comprised in the C-terminal amino acid sequence CPTGXQNYSP-COOH wherein X is Gly or Pro. The method according to claim 1,
Wherein said monoclonal antibody does not substantially recognize or bind to an elongated version of said C-terminal amino acid sequence,
Wherein said extended version is CPTGXQNYSPQZ-COOH, wherein Z is omitted or is one or more amino acids of collagen type III sequence. 3. The method according to claim 1 or 2,
Wherein said sandwich immunoassay method is used to quantify the amount of PIIINP cross-linked in a biological sample. The method of claim 3,
Further comprising correlating the amount of cross-linked PIIINP measured by said method with a standard sample of fibrosing disease for which the severity of illness is known, to assess the severity of the fibrotic disease. 5. The method of claim 4,
Wherein the fibrotic disease is liver disease. 6. The method according to any one of claims 3 to 5,
Wherein the biological sample is a biofluid. The method according to claim 6,
Wherein the biological fluid is serum, plasma, urine, amniotic fluid, tissue supernatant or cell supernatant. 8. The method according to any one of claims 1 to 7,
Wherein the sandwich immunoassay method is a radioimmunoassay, a fluorescent immunoassay, or an enzyme-linked immunoassay assay. 9. The method according to any one of claims 1 to 8,
Wherein said second monoclonal antibody is labeled. 10. The method of claim 9,
Wherein said second monoclonal antibody is an enzyme-linked antibody. 11. The method of claim 10,
Wherein the enzyme is horseradish peroxidase (HRP). 10. The method of claim 9,
Wherein the second monoclonal antibody is radiolabeled or the fluorophore is ligated. 9. The method according to any one of claims 1 to 8,
Wherein the second monoclonal antibody recognizes the second monoclonal antibody to determine the amount of binding of the second monoclonal antibody. A method for assessing the efficacy of an antagonist drug targeting lysyl oxidase (LOX)
Comprising quantifying the amount of PIIINP cross-linked in two or more biological samples using the sandwich immunoassay of claim 1,
Wherein the biological sample is obtained from the subject at a first time point and at one or more time points thereafter, during a period of administration of the antagonist drug to the subject,
Wherein a quantitative decrease in PIIINP cross-linked from said first time point to said one or more later time points during the period of administration of said antagonist drug means that said antagonist drug is an effective antagonist drug targeting LOX. 15. The method of claim 14,
Wherein the method evaluates the efficacy of an antagonist drug targeting LOXL2. A kit for use in a sandwich assay,
A solid support to which the first monoclonal antibody according to claim 1 is bound; And
A second monoclonal antibody according to claim 1,
Wherein said second monoclonal antibody comprises a labeling substance.

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