Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/08—Hepato-biliairy disorders other than hepatitis
  • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/70—Mechanisms involved in disease identification
  • G01N2800/7052—Fibrosis
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to a sandwich immunoassay for detecting in a biological sample cross-linked PIIINP, and its use in evaluating the efficacy of drugs targeting lysyl oxidases (LOXs).
• the invention also relates to a kit for performing the sandwich immunoassay.
• Fibrotic diseases are a leading cause of morbidity and mortality, e.g. cirrhosis with 800,000 deaths per year worldwide (1 ).
• a 'fibrotic disease' is any disease giving rise to fibrosis, whether as a main or a secondary symptom. Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli including persistent infections, autoimmune reactions, allergic responses, chemical insults, radiation, and tissue injury. Fibrosis is characterized by the accumulation and reorganization of the extracellular matrix (ECM). Despite having obvious etiological and clinical distinctions, most chronic fibrotic disorders have in common a persistent irritant that sustains the production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines, which together stimulate the deposition of connective tissue elements, especially collagens and proteoglycans, which progressively remodel and destroy normal tissue architecture (3,4).
• ECM extracellular matrix
• ECM Extracellular Matrix
• the ECM is a supramolecular structure with the ability to form aggregates of proteins, thus forming a dynamic scaffold linking cells together in a three dimensional network. This scaffold controls cell-matrix interactions and cell fate through up and down regulation of proteases (6).
• the ECM consists of collagens, laminins, proteoglycans, and other glycoproteins in various amounts and combinations, thereby providing a variety of biological components which can be modified by proteases to produce scaffolds with specific functions to meet the needs of the individual tissue (7).
• Collagen types I and III are the major structural proteins in the human body. Collagen type III is essential for collagen type I fibrillogenesis in the cardiovascular system and other organs (8,9). During fibrillar assembly the N-terminal propeptide of type III procollagen (which consists of three identical a-chains with a total molecular weight of 42 kDa) is cleaved off by specific N-proteases prior to incorporation of the mature collagen in the ECM. The cleaved propeptides may either be retained in the ECM or released into the circulation. However, the cleavage of the propeptide is sometimes incomplete, leaving the propeptide attached to the molecule.
• type III procollagen which consists of three identical a-chains with a total molecular weight of 42 kDa
• the cleaved propeptides may either be retained in the ECM or released into the circulation. However, the cleavage of the propeptide is sometimes incomplete, leaving the propeptide attached to the molecule.
• the level of the N- terminal propeptide of type III collagen (PIIINP) in a suitable sample can be a marker of formation and/or degradation of collagen type III.
• Remodeling of the ECM plays an important role in the pathogenesis of various diseases as altered components and non-coded modifications of the ECM leads to tissue stiffness and changes in the signaling potential of the intact ECM and fragments thereof.
• ECM remodeling is an important prerequisite for tissue function and repair, and is tightly controlled by the enzymes responsible for the synthesis and degradation of the ECM.
• PIMNP could be used as a biomarker for several fibrotic diseases, such as lung injury (14), viral and non-viral hepatitis (15), systemic sclerosis (16), vascular remodeling (17), and kidney diseases (18).
• Type III collagen is dominant in the early stages of fibrosis, while up-regulation of type I collagen is related to the later stages of fibrosis. Fibrosis occuring in the liver results in the deposition of collagen and release of propeptides, predominantly PIMNP. Consequently, PIMNP is one of the best studied markers for fibrogenesis (28,29,30).
• neo-epitopic fragment which is solely produced in the formation process (i.e. a fragment which is produced in the formation of collagen type III but not produced in the degradation of collagen type III).
• WO 2014/170312 discloses a monoclonal antibody which is specific for the C- terminal PIIINP neo-epitope comprised in the terminal amino acids of the C-terminal amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO:4), wherein X can be Gly or Pro.
• Bayer (33) discloses a sandwich ELISA which utilises a detector monoclonal antibody directed to the sequence H 2 N-GSPGPPGICQSCPTGPQNYSP-COOH (SEQ ID NO: 3), however the binding epitope is not defined.
• a specific sandwich immunoassay which utilises the neo-epitope specific antibody directed towards the C-terminal neo-epitope of PIIINP as disclosed in WO 2014/170312 may be useful in evaluating the efficacy of drugs that target lysyl oxidases (LOXs), particularly LOX antagonist drugs.
• LOXs lysyl oxidases
• Enzymatic collagen crosslinking by LOXs and processing of pro-collagens is key for tissue maturation and stability. In patients with organ fibrosis collagens become highly cross- linked, and thus are less prone to fibrosis resolution.
• LOXL2 a specific LOX
• novel LOXL2 antagonists are currently undergoing clinical trials.
• an assay that could be used to evaluate the efficacy of drugs targeting LOXs, such as LOX antagonists, would clearly be a useful tool for the pharmaceutical industry.
• the present invention is directed to a sandwich immunoassay for detecting in a biological sample cross-linked PIIINP where the cross-linked PIIINP comprises at least two strands of PIIINP joined together by inter-strand cross-linking.
• the method comprises contacting the biological sample comprising the cross-linked PIIINP with a first monoclonal antibody bound to a surface, where each strand of PIIINP comprised in the cross-linked PIIINP has a C-terminal neo-epitope of PIIINP generated by N-protease cleavage of intact type III procollagen, and adding a second monoclonal antibody.
• Both monoclonal antibodies are specifically reactive with the C-terminal neo-epitope of PIIINP, and said neo-epitope is comprised in a C-terminal amino acid sequence CPTGXQNYSP-COOH, where X is Gly or Pro.
• the method further comprises determining the amount of binding of the second monoclonal antibody.
• the present invention also is directed to a method for evaluating the efficacy of an antagonist drug targeting lysyl oxidases (LOXs).
• the method comprises using the sandwich immunoassay as described herein to quantify the amount of cross-linked PIIINP in at least two biological samples obtained from a subject at a first time point and at least one subsequent time point during a period of administration of the antagonist drug to the subject.
• a reduction in the quantity of cross-linked PIIINP from the first time point to the at least one subsequent time point during the period of administration of the antagonist drug is indicative of an efficacious antagonist drug targeting LOXs.
• the present invention is directed further to a kit for use in the sandwich immunoassay as described herein.
• the kit comprises a solid support to which is bound the first monoclonal antibody as described above and a labelled second monoclonal antibody as described herein.
• FIG. 1 Alignment of the targeted PIIINP a1 chain sequence in human (SEQ ID NO: 14) and rat (SEQ ID NO: 15) species (highlighted by the box). Position of the corresponding human (— ) and rat (— ) sequences within the alpha 1 chain of the N- terminal pro-peptide of type III collagen. The alignment was performed using the NLP CLUSTALW software.
• FIG. 2 Western Blot showing the specific bands of N-terminal propeptide of type III collagen in Amniotic fluid from a) rat and b) human recognized by the monoclonal antibody NB61 N62 (lane 1 and 3) and NB61 N62 + selection peptide (lane 2+4). Two bands around 52-60 kDA was observed for the rat, whereas one band was observed for human. Addition of selection peptide resulted in weakness of band intensity for both rat and human.
• FIGS. 3A-3D PRO-C3 ELISA runs showing typical calibration curves and native reactivity against human, rodent, and mouse material.
• FIG. 3A Calibration curve and inhibition of the competitive PRO-C3 ELISA using healthy human serum, plasma, and amniotic fluid (AF). The calibrator curve was diluted in 2-fold from 76.31 ng/mL, whereas native material was run diluted 1 :2 to 1 :16 as indicated (-).
• FIG. 3B Calibration curve and inhibition of the competitive PRO-C3 ELISA using healthy rat serum, plasma, and AF. The calibrator curve was diluted in 2-fold from 200 ng/mL, whereas native material was run undiluted to 1 :8 as indicated (-).
• FIG. 3A Calibration curve and inhibition of the competitive PRO-C3 ELISA using healthy human serum, plasma, and amniotic fluid (AF). The calibrator curve was diluted in 2-fold from 76.31 ng/mL, whereas native material was run diluted 1
• FIG. 3C Calibration curve and inhibition of the competitive PRO-C3 ELISA using healthy mouse serum and plasma.
• the calibrator curve was diluted in 2-fold from 200 ng/mL, whereas native material was run undiluted to 1 :4 as indicated (--).
• FIG. 3D Neo-epitope specificity of the PIIINP neo-epitope specific antibody using elongated peptide, i.e. peptide sequence of calibration peptide with one additional amino acid in the C-terminal end.
• the calibration curve, elongated peptide, and non-sense peptide were diluted in 2-fold from 76.31 ng/mL. The signal is seen as the optical density at 450 nm, subtracting the background at 650nm, as a function of peptide concentration.
• FIG. 4 Results of an in vitro model of lung fibroblasts ("scar-in-a-jar").
• FIG. 5 A comparison of Pro-C3X levels in extractions from keloids and extractions from normal skin.
• FIGS. 6A-6B Results of a study of patients with liver fibrosis.
• FIG. 7 Pictorial representation of the Pro-C3X assay.
• FIG. 8 Results of a study of patients with Alcoholic steatohepatitis.
• FIG. 9 Pro-C3X levels in supernatant collected at Day 10 from the Scar-in-a-Jar model. Significance was assessed by one-way ANOVA with Dunnett's multiple comparisons test comparing each condition with TGF- ⁇ alone. Data are shown as mean with SD. **** p ⁇ 0.0001.
• BAPN ⁇ -aminopropionitrile
• TGF- ⁇ transforming growth factor ⁇ -
• non-epitope refers to an N- or C-terminal peptide sequence at the extremity of a polypeptide, i.e. at the N- or C- terminal end of the of the polypeptide, and is not to be construed as meaning in the general direction thereof.
• competitive ELISA refers to a competitive enzyme-linked immunosorbent assay and is a technique known to the person skilled in the art.
• sandwich immunoassay refers to the use of at least two antibodies for the detection of an antigen in a sample, and is a technique known to the person skilled in the art.
• the monoclonal antibody NB61 N-62 refers to a neo- epitope specific antibody directed towards the C-terminal neo-epitope of PIIINP, said neo-epitope comprising the C-terminal sequence CPTGXQNYSP-COOH (SEQ ID NO:
• PRO-C3 is used to distinguish the herein described PIIINP assay from the PIIINP assays known in the art which are not based on the specific binding of neo-epitopes originating from PIIINP.
• PRO-C3X assay refers to the herein described sandwich immunoassay for detecting and quantifying cross-linked PIIINP.
• a monoclonal antibody suitable for use in the method of the invention was disclosed in WO 2014/170312 and is specifically reactive with a C-terminal neo-epitope of PIIINP, said neo-epitope being comprised in a C-terminal amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO:4), wherein X is Gly or Pro, and wherein said monoclonal antibody does not substantially recognise or bind an elongated version of said C-terminal amino acid sequence which is CPTGXQNYSPQZ-COOH (SEQ ID NO:
• the monoclonal antibody is specifically reactive with the neo-epitope C-terminal sequence CPTGPQNYSP-COOH (SEQ ID NO: 6) in human PIIINP, which is formed by the N-protease cleavage of PIMNP from intact procollagen type III at the Pro- Gin bond between amino acids P153-Q154 in human PIMNP.
• CPTGPQNYSP-COOH SEQ ID NO: 6
• the monoclonal antibody may be specifically reactive with the neo- epitope C-terminal sequence CPTGGQNYSP-COOH (SEQ ID NO: 7) in rodent PIMNP, which said neo-epitope is formed by the N-protease cleavage of PIMNP from intact procollagen type III at the Pro-Gin bond between amino acids P154-Q155 in rodent PIMNP.
• the ratio of the affinity of the monoclonal antibody for amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) to the affinity of said monoclonal antibody for elongated 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 , and most preferably at least 1 ,000,000 to 1.
• the monoclonal antibody does not recognise or bind a shortened version of a C-terminal neo-epitope of PIMNP, said shortened neo-epitope having the amino acid sequence CPTGXQNYS (SEQ ID NO: 8).
• the ratio of the affinity of the monoclonal antibody for amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) to the affinity of said monoclonal antibody for shortened 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 , and most preferably at least 1 ,000,000 to 1.
• the present invention relates to a sandwich immunoassay for detecting in a biological sample cross-linked PIIINP, said cross-linked PIIINP comprising at least two strands of PIIINP joined together by inter-strand cross-linking, said method comprising: contacting said biological sample comprising said cross-linked PIIINP with a first monoclonal antibody bound to a surface, wherein each strand of PIIINP comprised in the cross-linked PIIINP comprises a C-terminal neo-epitope of PIIINP generated by N- protease cleavage of intact type III procollagen;
• both said first monoclonal antibody and said second monoclonal antibody are specifically reactive with said C-terminal neo-epitope of PIIINP, said neo- epitope being comprised in a C-terminal amino acid sequence CPTGXQNYSP-COOH, wherein X is Gly or Pro.
• the monoclonal antibody does not substantially recognise or bind an elongated version of said C-terminal amino acid sequence which is CPTGXQNYSPQZ- COOH, wherein Z is absent or is one or more amino acids of the sequence of collagen type III.
• sandwich immunoassay uses the same antibody as both catcher and detector antibody, therefore a double strand peptide (i.e. cross-linked) can be recognized by the assay.
• the sandwich immunoassay is used to quantify the amount of cross- linked PIIINP in a biofluid, wherein said biofluid may be, but is not limited to, serum, plasma, urine, amniotic fluid, tissue supernatant or cell supernatant.
• the sandwich immunoassay may be, but is not limited to, a radioimmunoassay, fluorescence immunoassay, or an enzyme-linked immunosorbent assay.
• the second monoclonal antibody may be labeled in order to determine the amount of binding of said second monoclonal antibody.
• the second monoclonal antibody may be an enzyme-linked antibody.
• the enzyme may be, but is not limited to, horseradish peroxidase (HRP).
• the second monoclonal antibody may be radiolabeled or linked to a fluorophore.
• any suitable labeling system may be employed, such as, but not limited to, DNA reporters or electrochemiluminescent tags.
• a further labeled antibody which recognises the second monoclonal antibody may be used to determine the amount of binding of said second monoclonal antibody.
• the further labeled antibody may be labeled using a label as described above.
• the sandwich immunoassay may further comprise correlating the quantity of cross-linked PIIINP determined by said method with standard fibrotic disease samples of known disease severity to evaluate the severity of a fibrotic disease.
• a fibrotic disease may be, but is not limited to, liver disease.
• the sandwich immunoassay described herein may be used in a method for evaluating the efficacy of a drug targeting lysyl oxidases (LOXs), such as an antagonist drug targeting LOXs.
• LOXs drug targeting lysyl oxidases
• the present invention also relates to a method for evaluating the efficacy of an antagonist drug targeting lysyl oxidases (LOXs), wherein said method comprises using the sandwich immunoassay described herein to quantify the amount of cross-linked PIMNP in at least two biological samples, said biological samples having been obtained from a subject at a first time point and at least one subsequent time point during a period of administration of the antagonist drug to said subject, and wherein a reduction in the quantity of cross-linked PIMNP from said first time point to said at least one subsequent time point during the period of administration of the antagonist drug is indicative of an efficacious antagonist drug targeting LOXs.
• LOXs antagonist drug targeting lysyl oxidases
• the method quantifies the efficaciousness of the antagonist drug.
• the method evaluates the efficacy of an antagonist drug targeting LOXL2.
• the present invention relates to a kit for use in the sandwich immunoassay as described herein, the kit comprising a solid support to which is bound a first monoclonal antibody as described above; and a labelled second monoclonal antibody as described above.
• the sequence for the N-terminal propeptide of type III collagen was aligned between human, rat and mouse species and selected from homology between the species and uniqueness among other ECM proteins by protein blasting.
• the amino acid sequence 145'-CPTGPQNYSP-'153 (SEQ ID NO: 6) in the a1 chain PIIINP is 100% homologues between human and rat (FIG. 1 ).
• Generation of monoclonal antibodies was initiated by subcutaneous immunization of 4-5 week old Balb/C mice with 200 ⁇ emulsified antigen and 50 ⁇ g PIIINP neo-epitope C-terminal sequence (OVA-CGG- CPTGPQNYSP (SEQ ID NO: 10)) using Freund's incomplete adjuvant.
• the immunizations were repeated every 2 weeks until stable serum titer levels were reached.
• the mouse with the highest serum titer was selected for fusion.
• the mouse was rested for a month and then boosted intravenously with 50 ⁇ 9 PIIINP neo-epitope C-terminal sequence in 100 ⁇ 0.9% NaCI solution three days before isolation of the spleen.
• the spleen cells were fused with SP2/0 myeloma cells to produce hybridoma as described by (34), and cloned in culture dishes using the semi-medium method.
• the clones were plated into 96-well microtiter plates for further growth employing the limited dilution method to secure monoclonal growth.
• Native reactivity and affinity of the peptide were assessed using different biological materials such as urine, serum, and amniotic fluid (AF) from both humans and rats in a preliminary ELISA using 2 ng/ml biotinylated peptide on streptavidin-coated microtiter plates and the supernatants from growing monoclonal hybridoma cells.
• Human AF was obtained from 30 women undergoing elective lower segment Caesarean sections at the Beijing Obstetrics Gynecology Hospital over a 2 month period. 100-200 ml AF was collected directly after incision and the fluid was stored at - 20°C until use. The local ethical board had approved the study and all women provided written consent prior to collection.
• Rat AF was drawn from the uterus of pregnant Wistar rats two days prior to expected birth. Antibody specificity was tested in a preliminary assay using deselection and elongated peptides (i.e. calibrator peptide with ten amino acid substitutions and calibrator peptide with one additional amino acid at the cleavage site, respectively). The isotype of the monoclonal antibodies was determined using the Clonotyping System-HRP kit, cat. 5300-05 (Southern Biotech, Birmingham, AL, USA). Antibody characterization
• BCA Bicinchoninic acid
• rat or human AF was mixed with sample buffer (x2) and reducing agent (x10), heated at 70°C for 10 minutes, loaded on a 4-20% tris- glycein sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-page), and run for 1 hour at 180V. Protein bands were blotted onto a nitrocellulose membrane using the Invitrogen i-Blot gel transfer system according to manufacturer's instruction.
• the membrane was blocked in blocking buffer (5% skimmed milk in Tris-buffered saline with Tween (TBST) overnight at 4°C and incubated with 1 ⁇ g/ml horseradish peroxidase (HRP)-conjugated PIIINP neo-epitope specific monoclonal antibody NB61 N-62 for 2 hours.
• HRP horseradish peroxidase
• Specificity of the PIIINP neo-epitope specific monoclonal antibody was investigated by addition of excess PIIINP neo-epitope calibrator peptide and antibody in the ratio 10:1 and allowed to pre-incubate for 1 hour before it was added to the membrane for overnight incubation. After incubation the membranes was washed 4x10 minutes in TBST, incubated with 4 ml chemiluminescence detection kit (ECL), and developed using Amersham Hyperfilm.
• ECL ml chemiluminescence detection kit
• the subtype was determined to be an lgG1 subtype. From the Western Blot analysis it was seen that the PIIINP neo-epitope specific monoclonal antibody NB61 N- 62 recognized two bands with molecular sizes around 52-60 kDa in rat amniotic fluid, while only one band around 52 kDa was detected in human amniotic fluid. In addition, the signal could be partly inhibited by the selection peptide in the rat, and inhibited in human (FIG. 2). Native reactivity was observed using the NB61 N-62 antibody in the ELISA. Native reactivity was seen towards human serum, plasma, and AF as well as against rodent serum, plasma, and AF (FIGS. 3A-3C).
• the signal was slightly less inhibited against mouse serum and plasma.
• the signal of the competitive ELISA was inhibited using from 1 :2 to 1 :16, undiluted to 1 :8, or undiluted to 1 :4 in human, rodent, and mouse native material, respectively. Dilution of the native material approximately followed the same dilution pattern as the calibrator curve for all three species.
• Human AF inhibited the signal up to 100%; 80% for rat AF; 70% for human serum and plasma and rat serum; 44% for rat plasma, and 35% for mouse serum and plasma.
• the PRO-C3 competitive ELISA procedure was as follows: A 96-well streptavidin-coated ELISA plate from Roche, cat.1 1940279, was coated with the biotinylated peptide Biotin-CGG-CPTGPQNYSP (SEQ ID NO: 1 1 ) dissolved in coater buffer (50mM PBS-BTE + 10% sorbitol, pH 7.4), incubated for 30 min at 20°C in the dark and subsequently washed in washing buffer (20 mM Tris, 50 mM NaCI, pH 7.2).
• TMB tetramethylbenzinidine
• a 2-fold dilution of healthy serum and plasma samples from human and rats were used to determine linearity and calculated as percentage of recovery of the 100% sample.
• Antibody specificity was calculated as percentage of recovery of the 100% calibrator peptide (CPTGPQNYSP (SEQ ID NO: 6)), elongated peptide (CPTGPQNYSPQ (SEQ ID NO: 13)), and non-sense peptide (GSPGKDGVRG (SEQ ID NO: 12)).
• Lower limit of detection (LLOD) was calculated as the mean + 3xStandard Deviation (SD) of the blank from 21 determinations of standard K (i.e. buffer).
• Upper limit of detection (ULOD) was determined as the mean - 3xSD of 10 measurements of Standard A.
• LLOQ Lower limit of quantification
• the measurement range of the human PRO-C3 ELISA was determined by calculating ULOD and LLOQ providing a range from 0.867-60.1 ng/ml with a LLOD of 0.606 ng/ml.
• the technical performance of the PRO-C3 ELISA showed acceptable inter- and intra assay variation of mean 1 1.03% and 4.1 1 % (Table 1 ), with acceptance range below 15% and 10%, respectively.
• Table 1 Inter- and intra-assay variation for the PRO-C3 assay using human serum quality control samples # 1 -8 (HS1 - HS8). The variation was calculated as the mean variation between 10 individual determinations of each sample.
• Dilution recovery was performed using healthy serum and plasma samples from humans, rat and mouse. The dilution recovery was within the acceptable 100 ⁇ 20% recovery (Table 2). Further dilution resulted in measurements below LLOQ.
• Table 2 Percentage dilution recovery for the PRO-C3 assay using human-, rat-, and mouse samples.
• Table 3 Spiking recovery of calibrator peptide in human serum or plasma, and human AF in human serum or plasma. The recovery was calculated as percent recovery of calculated peptide/AF in serum/plasma compared to pure serum/plasma.
• Concentration of calibrator peptide were 38.16 ng/ml (StdB), 19.08 ng/ml (StdC), 9.54 ng/ml (StdD), 4.77 ng/ml (StdE), 2.39 ng/ml (StdF) and 1.19 ng/ml (StdG).
• AF was added in 2-fold dilution starting from 1 :2.
• the stability of the analyte was acceptable up to four freeze/thaw cycles with
• each of the sequences are synthesized and used as calibrator peptides in the PRO-C3 ELISA as described in example 2.
• the resultant calibration curves are used to determine the IC 50 values of each sequence/antibody combination.
• the ratio of ICso[target] / ICso[elongated or shortened] defines the ratio of binding affinity.
• enzymatic collagen crosslinking by lysyl oxidases (LOXs) and processing of pro-collagens is key for tissue maturation and stability.
• monitoring inter-strand cross-linking of pro-collagen type III prior to enzymatic processing may prove useful for monitoring in-vivo activity of LOXs.
• This can be achieved by detecting and quantifying cross-linked PIIINP (i.e. two or more strands of PIIINP bound together by inter-strand links formed by LOXs in pro-collagen type III prior to enzymatic processing of the pro-collagen).
• a higher level of cross-linked PIIINP detected in the circulation would be indicative of greater LOX activity.
• monitoring the level of cross-linked PIIINP during drug trials for drugs targeting LOX such as LOX antagonists, could provide useful efficacy data for said drugs.
• Streptavidin-coated plates were coated with 100 ⁇ /well of 1 ⁇ g/ml biotinylated catcher antibody (biotin-linked NB61 -N62) and incubated at 20°C, 300 rpm shaking for 30 minutes. Plates were washed five times in washing buffer (20 nM TRIS, 50 mM NaCI, pH 7.2). Sample, standard or control (20 ⁇ ) was added and followed immediately by addition of 100 ⁇ assay buffer and incubated at 4°C, 300 rpm shaking for 20 hours. After incubation, plates were washed five times in washing buffer.
• Pro-C3X was significantly elevated in later stages of disease, when fibrosis is more severe, with levels similar to healthy controls in early stages of disease (FIG. 6A). In comparison, Pro-C3 levels differed at all stages of the disease (FIG. 6B).
• FIG. 7 shows the Pro-C3X assay and provides a pictorial explanation for the reasoning behind this conclusion:
• Pro-C3X assay if cross linked PIIINP is present then the first antibody will bind to the free epitope on a first strand of PIIINP and subsequently the second antibody will bind to the free epitope on a second strand of PIIINP. However, if non- cross-linked PIIINP is present then the surface-bound antibody will bind to the free epitope of the non-cross linked collagen type III, but the second antibody will fail to bind as the binding epitope is already occupied, therefore addition of the second antibody will fail to produce a signal. Thus, the signal from the Pro-C3X assay is exclusively due to the detection of cross linked PIIINP.
• the signal obtained from the Pro-C3 assay is an aggregate signal of cross-linked and non-cross-linked PIIINP.
• ProC3 only correlated negatively with albumine, while ProC3X correlated with albumine, bilirubine and Gamma-Glutamyl Transpeptidase (GGT) (FIG. 8).
• GTT Gamma-Glutamyl Transpeptidase
• the late stage increase in Pro-C3X suggests increase in cross-linking of PIIINP, which would be in accordance with increased scarring of the liver (i.e. increased LOX activity).
• Fibrosis is the accumulation of extracellular matrix (ECM) within affected tissues, which can lead to organ failure and ultimately death.
• ECM extracellular matrix
• TGF transforming growth factor
• fibroblasts are the main cell type responsible for the excessive accumulation of ECM proteins, especially collagens.
• Scar-in-a-Jar known to generate ECM and cross-links
• Pro-C3X ELISA Pro-C3X ELISA
• Healthy human lung fibroblasts (L248) were grown to confluence, after which they were seeded at a density of 30,000 cells/well.
• the cells were grown in DMEM media containing 0.4% FCS, 225 mg/mL ficoll 70, 150 mg/mL ficoll 400, and 1 % ascorbic acid for 18 days.
• the cells were stimulated with 1 ng/mL TGF- ⁇ with or without the lysyl oxidase (LOX) inhibitor ⁇ -aminopropionitrile (BAPN; 0.02 or 0.2 mM) to inhibit formation of cross-links.
• LOX lysyl oxidase
• BAPN lysyl oxidase
• Unstimulated cells or cells grown in media not containing ficoll were used as controls. Media was changed on day 3, 6, 10, and 14.
• Fibroblast viability was assessed using the AlamarBlue assay.
• Pro-C3X levels were assessed in the collected supernatant, using the Pro-C3X sandwich ELISA described above. Results.
• TGF- ⁇ stimulation induced the release of Pro-C3X from day 3 with a peak in Pro-C3X levels at day 10 showing a 14-fold increase as compared with unstimulated cells (p ⁇ 0.0001 ; Figure 9).
• Treatment with 0.02 mM BAPN had no significant effect, but 0.2 mM BAPN induced a significant decrease in Pro-C3X levels as compared with TGF- ⁇ stimulation only (0.59-fold change, pO.0001 ; Figure 9).
• the pan LOX inhibitor BAPN significantly reduced Pro-C3X levels at a concentration of 0.2 mM, indicating that the Pro-C3X ELISA assesses a cross-linked epitope.
• the Pro-C3X ELISA can be used to evaluate fibroblast activity and thus be used to screen potential anti-fibrotic compounds.
• TGF- ⁇ stimulation induced the release of Pro-C3X, a marker of the cross-linked collagen type III pro-peptide.
• the Pro-C3X assay is a second generation assay combining the cleavage neo-epitope of the collagen type III pro-peptide and the presence of cross- linking in the molecule.
• This assay is therefore providing additional information to the measurement of Pro-C3, because it describes a different process in the fibrosis timeline; that is, the cross-linking of collagen molecules in the scar.
• the Pro-C3X assay can therefore be used to test the efficacy of drugs targeting LOX, particularly LOX antagonists/inhibitors, since the use of a LOX inhibitor was shown to reduce the presence of the cross-linked PIIINP biomarker.
• TIMP-3 inhibits the procollagen N- proteinase ADAMTS-2. Biochem J 2006; 398(3): 515-519.

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