Elastin Assay

Abstract

Described herein are monoclonal antibodies, assay kits and immumoassay methods for quantifying elastin fragments having a C-terminus amino acid sequence LPGGYGLPYT (SEQ ID NO: 1) in a patient biofluid sample, and uses thereof for detecting or quantifying fibrotic diseases such as chronic obstructive lung disease (COPD).

Claims

1. An immunoassay method for quantifying peptides having a C-terminus amino acid sequence LPGGYGLPYT (SEQ ID NO: 1) in a patient biofluid sample, said method comprising contacting said patient biofluid sample with a monoclonal antibody specifically reactive with said C-terminus amino acid sequence LPGGYGLPYT (SEQ ID NO: 1), and determining the amount of binding between said monoclonal antibody and said C-terminus amino acid sequence.

2. The method of claim 1, wherein the monoclonal antibody does not specifically recognise or bind a C-extended elongated version of said C-terminus amino acid sequence or a C-truncated shortened version of said C-terminus amino acid sequence.

3. The method of claim 1, wherein the patient biofluid sample is blood, urine, synovial fluid, serum, BALF, or plasma.

4. A method of immunoassay for detecting or quantifying a fibrotic disease in a patient, the method comprising contacting a patient biofluid sample with a monoclonal antibody specifically reactive with a C-terminus amino acid sequence LPGGYGLPYT (SEQ ID NO: 1), determining the amount of binding between said monoclonal antibody and peptides comprising said C-terminus amino acid sequence, and correlating said amount of binding with i) values associated with normal healthy subjects and/or ii) values associated with known fibrotic disease severity and/or iii) values obtained from said patient at a previous time point and/or iv) a predetermined cut-off value.

5. A method as claimed in claim 4, wherein the fibrotic disease is chronic obstructive pulmonary disease (COPD).

6. A method as claimed in claim 4, wherein the predetermined cut-off value is at least 150.0 ng/mL.

7. The method of claim 4, wherein the monoclonal antibody does not specifically recognise or bind a C-extended elongated version of said C-terminus amino acid sequence or a C-truncated shortened version of said C-terminus amino acid sequence.

8. The method of claim 4, wherein the patient biofluid sample is blood, urine, synovial fluid, serum, BALF, or plasma.

9. A method for determining whether a patient is responding positively to a treatment for a fibrotic disease, wherein said method comprises using the method of claim 1 to quantify the amount of peptides comprising the C-terminus amino acid sequence LPGGYGLPYT (SEQ ID NO: 1) in at least two patient biofluid samples, said patient biofluid samples having been obtained from said patient at a first time point and at at least one subsequent time point during a period of administration of the treatment to said patient, and wherein a reduction in the quantity of peptides comprising the C-terminus amino acid sequence LPGGYGLPYT (SEQ ID NO: 1) from said first time point to said at least one subsequent time point during the period of treatment is indicative of said patient responding positively to said treatment.

10. The method of claim 9, wherein the fibrotic disease is COPD.

11. A monoclonal antibody specifically reactive with a C-terminus amino acid sequence LPGGYGLPYT (SEQ ID NO: 1).

12. An assay kit comprising a monoclonal antibody specifically reactive with a C-terminus amino acid sequence LPGGYGLPYT (SEQ ID NO: 1), and at least one of: a streptavidin coated well plate; a biotinylated peptide Biotin-L-LPGGYGLPYT (SEQ ID NO: 5), wherein L is an optional linker; a secondary antibody for use in a sandwich immunoassay; a calibrator peptide comprising the C-terminus amino acid sequence LPGGYGLPYT (SEQ ID NO: 1) an antibody biotinylation kit; an antibody HRP labeling kit; and an antibody radiolabeling kit.

13. An assay kit as claimed in claim 12, wherein the monoclonal antibody is raised against a synthetic peptide having the amino acid sequence LPGGYGLPYT (SEQ ID NO: 1).

Description

FIGURES

[0032] FIG. 1. Measurement of ELP-3 in healthy subjects and in patients with COPD.

EXAMPLES

[0033] The presently disclosed embodiments are described in the following Examples, which are set forth to aid in the understanding of the disclosure, and should not be construed to limit in any way the scope of the disclosure as defined in the claims which 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 present disclosure nor are they intended to represent that the experiments below are all or the 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 indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0034] In the following examples, the following materials and methods were employed.

[0035] Reagents

[0036] All applied reagents used in the presented experiments were high quality chemicals from Sigma Aldrich (St. Louis, Mo., USA) and Merck (Whitehouse Station, N.J., USA). The 96-well streptavidin-coated microtiterplates used were from Roche, Basel, Switzerland. The assay buffer applied consisted of 50 mM Tris-buffered saline (TBS) 2 g NaCl, pH 8.0. 3,3,5,5-Tetramethylbenzidine (TMB) was from Kem-EN-Tec Diagnostics. The synthetic peptides used for immunization and assay development were 1) Immunogenic peptide: KLH-CGG-LPGGYGLPYT (SEQ ID NO: 4), Biotinylated peptide: Biotin-LPGGYGLPYT (SEQ ID NO: 5), 3) Standard peptide: LPGGYGLPYT (SEQ ID NO: 1), and 4) Elongated peptide: LPGGYGLPYTT (SEQ ID NO: 2). All synthetic peptides were purchased from American peptide Company, Sunnyvale, Calif. USA. Reagents applied for in vitro cleavages: Elastin (Sigma, E7152) Human neutrophil elastase (HNE) protein (Abcam, ab91099), PR3 (EPC ML734), and complete protease inhibitor (Roche 1186153001).

[0037] Selection of Target Sequence

[0038] Cleavage sites specific for PR3 in elastin (Uniprot: P15502-ELN_Human) have previously been identified by mass spectrometry (12). The PR3 generated elastin decapeptides were blasted for homology to decapeptide sequences from other proteins using the NPS@: network protein sequence analysis. Based on this the sequence LPGGYGLPYT (SEQ ID NO: 1) was selected for the ELP-3 assay antibody development. The presence of the identified sequence was validated by mass spectrometry in-house analysis of PR3 cleavage of elastin in vitro.

[0039] Antibody Development

[0040] Immunizations were performed in 6 mice, for each selected target sequence, that were 4-6 weeks old using Freund's incomplete adjuvant (KLH-CGG-LPGGYGLPYT (SEQ ID NO: 4)) subcutaneously in the abdomen with 200 ul emulsified antigen (50 ug per immunization). The four initial immunizations were performed every second week followed by additional four immunizations performed every fourth week. The mouse with the highest serum titer was selected for fusion. The mouse was rested for a month and then boosted intravenously with 50 ul immunogenic peptide in 100 ul 0.9% NaCl solution three days before isolation of the spleen. The spleen cells were fused with SP2/o myeloma cells to produce hybridroma as described by Gefter et al and cloned in culture dishes (13). The clones were plated into 96-well microtiter plates for further growth employing the limited dilution method to ensure monoclonal growth. Supernatants of antibody-producing hybridoma were screened for reactivity against standard peptide and native material in an indirect ELISA using streptavidin-coated plates. Biotin-LPGGYGLPYT (SEQ ID NO: 5) was used as screening peptide, while standard peptide LPGGYGLPYT (SEQ ID NO: 1) was used as a calibrator to test for further specificity of clones.

[0041] ELISA Protocol

[0042] Supernatant from a selected clone for the targeted sequence was collected and monoclonal antibody was purified using HiTrap affinity columns (GE Healthcare Life Science, Little Chalfront, Buckinghamshire, UK).

[0043] Based on the collected antibodies the ELP-3 assay was developed as competitive ELISA assay. A 96-well streptavidin-coated microtiter plate (Roche, Basel, Switzerland) was coated with 100 ul biotinylated screening peptide (Biotin-LPGGYGLPYT (SEQ ID NO: 5)) dissolved in assay buffer for 30 min at 20 C. with shaking. Plates were washed five times in washing buffer (20 mM TRIS, 50 mM NaCl, pH 7). Sample/standard/control (20 ul) was added and followed by addition of (100 ul) monoclonal antibody and incubated for 3 h at 4 C. with shaking. After incubation, plates were washed five times in washing buffer. A volume of 100 ul of secondary HRP labeled goat anti-mouse antibody was added followed by 1 h incubation at 20 C. with shaking. Plates were then washed and TMB was added and the plate was analyzed by a SpectraMax M reader (Molecular Devices, Ca, USA) at 450 nm with 650 nm as the reference. A standard curve was plotted using a 4-parametric mathematical fit model. Each ELISA plate included kit controls to monitor inter-assay variation. All samples were measured within the range of the assay. All samples below the level of lower limit of quantification (LLOQ) were assigned the value of LLOQ.

[0044] Technical Evaluation

[0045] To determine linearity of dilution healthy human serum (n=4), heparin plasma (n=4), citrate plasma (n=4) and EDTA plasma was applied. Antibody specificity was calculated as percentage of signal inhibition of 2-fold diluted standard peptide (LPGGYGLPYT (SEQ ID NO: 1)), elongated peptide (LPGGYGLPYTT (SEQ ID NO: 2)), non-sense peptide (PGGVPGGVFY (SEQ ID NO: 6)) and non-sense coater (LPGGYGLPYT-K-Biotin (SEQ ID NO: 7)). The linarites were assessed by the percentage recovery of undiluted samples. The intra and inter-assay variation was determined by 10 independent measurements of 7 quality control samples run in double determinations. Lower limit of detection (LLOD) was determined as mean+3standard deviations (SD) of 21 blank (buffer) samples. Upper limit of detection (ULOD) was determined as the mean3SD measurements of standard A. Lower limit of quantification (LLOQ) was determined as the lowest concentration measured in human serum with an error lower than 30%. The accuracy was measured in healthy human serum samples spiked with standard peptide and in serum, and then calculated as the percentage recovery of spiked peptide or serum in buffer. The assay was tested for interference by calculating the percentage recovery of analyte in non-spiked healthy serum compared to human serum spiked with lipids (low=4.83 mM, high=10.98 mM), hemoglobin (low=0.155 mM, high=0.310 mM), and biotin (low=30 ng/ml, high=90 ng/ml).

[0046] Analyte Stability

[0047] Analyte stability was evaluated by incubation of three healthy human serum and plasma samples at either 4 or 20 C. for 2, 4 and 24 hours and calculated as the percentage of the samples kept at 20 C (0 hour sample). Additionally, the freeze thaw stability was determined for three healthy human serum and plasma samples to up to four freeze and thaw.

[0048] Cleavage Analysis

[0049] In order to generate the ELP-3 fragment in vitro, elastin was incubated for 2, 4, 24 or 48 hours at 37 C. with PR3 or HNE. Elastin was reconstituted in buffer (Tris-HCl, 150 mM NaCl, pH 7.5) for a concentration of lmg/ml. Cleavage solutions contained a final concentration of 100 ug/ml elastin and 1 ug/ml protease for the PR3 and 100 ug/ml elastin and 2 ug/ml protease for the HNE cleavage (buffer: Tris-HCl, 150 mM NaCl, pH 7.5). All solutions were immediately frozen until analysis.

[0050] Ethics Statement

[0051] The work performed in mice was approved by the National Authority (The Animal Experiments inspectorate, approval number: 2013-15-2934-00956). All mice were treated according to the guidelines for animal welfare.

[0052] The assessment of ELP-3 in COPD patients was performed in a cohort previously described by Sand et al. (14). The study included 68 participants and complies with the Declaration of Helsinki and Good clinical practice Guidelines, was approved by the local ethics committee (protocol number H-6-2013-014). All participants provided informed consent before all study-related assessments.

[0053] Inclusion criteria were a COPD diagnosis and FEV1 <80% of predicted value. Exclusion criterion was an acute exacerbation of COPD leading to hospitalization within four weeks prior to the study. ELP-3 levels in the COPD patients were compared to the levels of commercially available control sera from healthy donors (Valley Bio-Medial, Winchester, Va., USA). All measurements were performed blinded.

[0054] Statistical Analyses

[0055] Differences in ELP-3 levels between healthy controls and COPD patients were assed using a two-tailed Mann-Whitney unpaired t-test. Comparision of age in healthy controls vs COPD patients was performed using the Mann-Whitney unpaired t-test. Differences were considered statistically significant if p<0.05. Asterisks indicate the following: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Graphs are shown as MeanStandard error of the mean. All statistical analyses were performed in GraphPad Prism v. 7 (Graph Pad Software, La Jolla, Calif., USA) or MedCalc (Ostend, Belgium).

[0056] Results

[0057] Clone Characterization

[0058] Supernatants from antibody producing hybridomas were screened for reactivity against standard peptide, elongated peptide, and truncated peptide in an indirect ELISA. The peptide Biotin-LPGGYGLPYT was used to screen for reactivity. The antibody clone NBH116/6A10-E7-D12 was selected for ELP-3 and antibodies were purified from the supernatant. Based on this the competitive ELISA assay was established. Native reactivity was observed in serum and plasma, and no inhibition was observed using the elongated peptide or non-sense peptide.

[0059] Technical Evaluation

[0060] The measurement was determined based on the linear range of the standard curve. The mean intra- and inter-assay variation ranged from 2.4-10% and 4.7-14.5%, respectively.

[0061] Linearity of dilution was observed when diluted 1+3 for human serum and citrate plasma (but not EDTA and heparin plasma). Serum samples were spiked with peptide or another serum sample in seven different concentrations. The spiking recovery for both serum and peptides were within an acceptable range. Mean recovery % values were within 20% in reference with expected concentration of spiked samples and therefore acceptable. Analyte stability was acceptable during short term storage (up to 48 hours) at 4 and 20 C., during freeze/cycles and neither hemoglobin, lipids or biotin reduced the signal for any of the two assays.

[0062] In Vitro AnalysisELP-3 is Generated by PR3

[0063] Elastin was incubated with different elastases in order to determine whether ELP-3 was specific for proteinase 3 cleavage. ELP-3 was generated by elastin cleaved by PR3 after 2-48 h. Detectable levels of fragments recognized by ELP-3 were generated by HNE cleavage, but in much lower concentrations compared to the respective assay. No cross reactivity was observed towards the intact elastin, buffer control or protease controls for the assay.

[0064] ELP-3 Levels were Increased in COPD as Compared to Healthy Controls

[0065] To determine whether ELP-3 levels are increased in COPD, ELP-3 was assessed in a previously described cohort of 68 patients with clinically stable COPD. Of these, 26 also attended a follow-up visit after four weeks. Twenty two samples from healthy donors were included for comparison. Demographics can be seen in Table 1 and have previously been described by Sand et al. (14).

TABLE-US-00001 TABLE 1 Patient demographics. Mean (SD) COPD Healthy control (n = 68) (n = 26) p-value Age, Mean (SD) 71.1 (9.0) <0.0001 Male, n (%) 40 (59) 0.52 BMI, Mean (SD) 24.5 (6.1) NA FEV1 % of 40.4 (16.3) NA predicted value FEV/FVC ratio % 49.6 (0.14) NA GOLD 2/3/4, % 34%/34%/32% NA ELP-3 (ng/ml), 217.9 (168.6- 38.4 (33.8- p < 0.0001 Median (95% CI) 255.0) 42.3)

[0066] As demonstrated in FIG. 1, ELP-3 is statistically elevated in COPD patients when compared to healthy subjects (p<0.0001).

[0067] In this specification, unless expressly otherwise indicated, the word or is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator exclusive or which requires that only one of the conditions is met. The word comprising is used in the sense of including rather than in to mean consisting of. All prior teachings acknowledged above are hereby incorporated by reference.

REFERENCES

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