Detection of cleavage activity of an enzyme

Abstract

The present invention relates to detecting cleavage activity of an enzyme. The various aspects of the invention include an enzyme detection device, kit, method and use for detecting or measuring the presence in a test sample of the activity of an enzyme capable of cleaving a substrate. The invention also relates to indicator and binding molecules useful for carrying out the invention. The enzyme substrate contains a hidden binding site which is only revealed upon cleavage by the enzyme.

Claims

1. An enzyme detection kit for detecting the presence of cleavage activity of an enzyme, the kit comprising: (i) an intact indicator molecule, the intact indicator molecule comprising (a) a cleavage region comprising at least one cleavage site that is specific for the enzyme, the cleavage region defining an end of a detectable fragment or cleavage part of the indicator molecule when the intact indicator molecule is cleaved at the cleavage site, the detectable fragment or cleavage part of the indicator molecule comprising a binding site, wherein the binding site of the detectable fragment or cleavage part of the indicator molecule is a conformational epitope that is not formed in the intact indicator molecule; and (b) a capture site which is present in the intact indicator molecule and is present in the detectable fragment or cleavage part of the indicator molecule; (ii) capture molecules which are (a) capable of binding to the capture site of the intact indicator molecule and (b) capable of binding to the capture site of the detectable fragment or cleavage part of the indicator molecule; (iii) a solid support to which the capture molecules are attachable or attached to form a capture zone; and (iv) binding molecules which are (a) capable of binding to the binding site of the detectable fragment or cleavage part of the indicator molecule and (b) incapable of binding to the intact indicator molecule.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will now be described by way of example with respect to the accompanying drawings in which:

(2) FIG. 1 is a schematic view of four different formats of the assay in accordance with the invention. Each format relies upon the same basic components of solid support (1), capture molecule (2), an indicator molecule containing a capture site (3) and a cleavage site (4) and a binding molecule (5) that binds to the indicator molecule only after cleavage (6) has occurred.

(3) FIG. 2 is a schematic view of an enzyme detection device in accordance with the present invention and shows operation of the device in the absence (FIG. 2A) or presence (FIG. 2B) of enzyme cleavage activity.

(4) FIG. 3 shows the visual read-out of the assay (shown in FIG. 2) as levels of MMP activity in the test sample are increased.

(5) FIG. 4 is a schematic view of an enzyme detection device in accordance with the present invention. The figure specifies the exact longitudinal dimensions and position of each of the card components.

(6) FIG. 5 shows an example of synthesis of a structurally constrained indicator molecule.

(7) In FIG. 5A initially, a linear peptide (1) is synthesised, for example using solid phase Fmoc chemistry. The peptide may be purified for example by High Performance Liquid Chromatography (HPLC). The peptide is then constrained, or cyclised, by reaction between thiol groups on the peptide (2) and the scaffold molecule (3). This reaction produces a structurally constrained clipped peptide (4).

(8) In FIG. 5B, the indicator molecule is synthesised to include the capture site (1), for example by synthesis of the linear peptide on a pre-loaded Biotin-PEG resin.

(9) FIG. 6 shows schematically the ability of the binding molecules used in the invention to bind exclusively to the cleaved indicator molecule. In the absence of enzyme cleavage activity, the structurally constrained indicator molecule (1) is not bound by the antibody binding molecule (2). This antibody is generated using the cleaved indicator molecule (3) as antigen and thus only binds to this open form of the molecule.

(10) FIG. 7 (FIGS. 7A and 7B) demonstrates the sensitivity of the assay of the invention when run with spiked MMP-9 buffer samples. The detectable limit for MMP-9 was approximately 4 ng/ml with a sample volume of 75111. FIG. 7A shows reader values across the entire concentration range of MMP-9, whereas FIG. 7B is an expanded view at MMP-9 concentrations between 0 and 15 ng/ml.

(11) FIG. 8 demonstrates that the specific version of the assay of the invention uses a cleavable sequence that is biased towards MMP13, MMP12, MMP9, MMP8 and MMP2. Other versions of the assays of the invention may use sequences with different targets depending on the application required.

(12) FIG. 9 demonstrates that measurable amounts of active proteases (in particular MMPs, including MMP-9) can be found in urine samples and that higher levels are present in samples obtained from patients with a respiratory disease. A significant difference was observed with COPD samples when compared to samples collected from healthy controls (P=0.03) and CF samples to healthy controls (P=0.01).

(13) FIG. 10 is a graph comparing the ability of the assay to detect MMP activity in the presence or absence of EDTA. The graph shows that addition of EDTA to the wound samples inhibits the readout, confirming the presence of MMP in the samples and also confirming that the assay is specifically measuring active MMPs.

(14) FIG. 11 contains graphs (FIGS. 11A and 11B) comparing the ability of a commercially available active MMP-9 assay kit and the assay of the invention to detect MMP9. FIG. 11A shows reader values across the entire concentration range of MMP-9, whereas FIG. 11B is an expanded view at MMP-9 concentrations between 0 and 50 ng/ml. Both figures demonstrate that the method of the invention produced a steeper curve. According to both assays, colour development as shown by the absorbance values was seen at 4 ng/ml MMP9, the lowest standard tested.

(15) FIG. 12 shows MMP9 standard curves using ELISA and lateral flow embodiments of the invention.

(16) FIG. 13 shows a number of scaffold molecules useful in the indicator molecules described herein.

(17) FIG. 14 shows a number of scaffold molecules useful in the indicator molecules described herein, together with proposed nomenclature.

(18) FIG. 15 shows some attachment options for scaffold molecules to the indicator molecules. FIG. 15A shows products of cleavage at a single cleavage site and FIG. 15B shows products of cleavage at two separate cleavage sites.

(19) FIG. 16 shows analytical HPLC of the MOL386 peptide.

(20) FIG. 17 is a mass spectrum of the MOL386 peptide.

(21) FIG. 18 is a mass spectrum of the MOL386 peptide modified with PEG-biotin.

(22) FIG. 19 is a mass spectrum analysis of the cyclised MOL386 peptide.

(23) FIG. 20 is a mass spectrum analysis of the cyclised MOL386 peptide modified with PEG-biotin.

(24) FIG. 21 shows MMP9 standard curves for all combinations shown in FIG. 1.

(25) FIG. 22 presents the performance of the best combinations derived from the results shown in FIG. 21.

(26) FIG. 23 shows a cyclised peptide substrate for Human Neutrophil Elastase (HNE) in both pre-digested and digested forms.

(27) FIG. 24 shows HPLC analysis of elastase digestion of a cyclised peptide substrate including the amino acid sequence of SEQ ID NO: 3. FIG. 24A presents the raw plot data. FIG. 24B presents a time course showing the relative increase in product and decrease in substrate over time.

(28) FIG. 25 presents mass spectrometric data confirming that the cyclised SEQ ID NO: 3 substrate is cleaved at a single site. FIG. 25A is the substrate plot and FIG. 25B is the hydrolysed product.

(29) FIG. 26 presents one route for generating an immunogen to raise antibodies specific for the cleaved form of the indicator molecule shown in FIG. 23.

(30) FIG. 27 presents an alternative route for generating an immunogen to raise antibodies specific for the cleaved form of the indicator molecule shown in FIG. 23.

(31) FIG. 28 shows mass spectrometric characterisation of the MOL488 peptide.

(32) FIG. 29 shows mass spectrometric characterisation of the MOL488 peptide following attachment to the scaffold molecule (derived from 1,3-dibromomethylbenzene).

(33) FIG. 30 shows mass spectrometric characterisation of the cyclised MOL488 peptide (i.e. attached to the scaffold) following cleavage using HNE.

DESCRIPTION OF PREFERRED EMBODIMENTS

(34) FIG. 1 is a schematic view of four different formats of the assay in accordance with the invention. Each format relies upon the same basic components of solid support (1), capture molecule (2), an indicator molecule containing a capture site (3) and a cleavage site (4) and a binding molecule (5) that binds to the indicator molecule only after cleavage (6) has occurred.

(35) In formats 1 and 4, the capture molecule (2) is streptavidin. Here, the capture molecule (2) binds to a biotin capture site (3) within the indicator molecule. In formats 2 and 3, the capture molecule (2) is an antibody. Here, the capture molecule (2) binds to an epitope capture site (3) within the indicator molecule. The epitope is found in the alternative long peptide (ALP) which is derived from human chorionic gonadotropin (hCG).

(36) Once the indicator molecule of the invention is added to a test sample, any enzyme specifically recognising the cleavage site (4) present, may cleave the indicator molecule (6). This cleavage event (6) produces a binding site for the specific antibody binding molecule (5). The binding molecule (5) is unable to bind to the indicator molecule until cleavage (6) has occurred. Thus, in formats 1 and 3 the antibody binding molecule (5) binds to the amino acid sequence GPQG produced as a result of cleavage of the GPQGIFGQ sequence. In formats 2 and 4, on the other hand, the antibody binding molecule (5) binds to the amino acid sequence QGFI, also produced as a result of cleavage of the GPQGIFGQ sequence. In each format, the antibody binding molecule (5) does not bind to the GPQGIFGQ sequence prior to cleavage (not shown).

(37) FIG. 2 is a schematic view of an enzyme detection device in accordance with the present invention and shows operation of the device in the absence (FIG. 2A) or presence (FIG. 2B) of enzyme cleavage activity. The test strip includes an adhesive liner (1) upon which the other components of the device are assembled. From right to left, the sample application zone (2) is in the form of an absorbent pad. This is laid partially overlapping the conjugate pad (3), which is impregnated with the labelled binding molecules (7). In alternative embodiments, the labelled binding molecules may be impregnated in the sample application zone and this removes the need for a separate conjugate pad. The conjugate pad (3) is in fluid connection with a nitrocellulose membrane (4). The nitrocellulose membrane (4) contains immobilized streptavidin molecules (5) which define a capture zone. The membrane (4) further contains immobilized further binding molecules (6) downstream of the capture zone which bind to further labelled molecules (11) which pass through the device with the sample and form a separate control zone. Alternatively, the immobilised further binding molecules may bind to labelled binding molecules (7). The device optionally further comprises an absorbent pad (8) to absorb any test sample and reagents reaching the end of the device.

(38) In use, the indicator molecule (9) is added to the test sample prior to bringing the test sample into contact with the sample application zone (8) of the device. As shown in FIG. 2A, in the absence of enzyme cleavage activity in the test sample, the indicator molecule (9) remains uncleaved at the cleavage site. Upon sample flow into the conjugate pad (3), the binding molecules (7) are unable to bind to the indicator molecule (9) because cleavage of the cleavage site has not occurred. The indicator molecules become bound at the capture zone via the interaction between streptavidin (5) and the biotin capture site (10) of the indicator molecule (9). The labelled binding molecules (7) are not immobilized at the capture zone because they cannot bind to the indicator molecules (9). Accordingly, the labelled binding molecules flow through to the control zone and beyond. Further labelled molecules (11) also pass through the device to the control zone where they are immobilized by binding to the immobilized further binding molecules (6). Thus, absence of enzyme cleavage activity is displayed as a signal only at the control zone, but not at the capture zone. Excess sample, potentially containing labelled binding molecules (7), flows into the absorbent pad (8).

(39) As shown in FIG. 2B, in the presence of enzyme cleavage activity in the test sample, the indicator molecule (9) is cleaved at the cleavage site. Upon sample flow into the conjugate pad (3), the binding molecules (7) are able to bind to the indicator molecule (9) because cleavage of the cleavage site has occurred. The indicator molecules become bound at the capture zone via the interaction between streptavidin (5) and the biotin capture site (10) of the indicator molecule (9). The labelled binding molecules (7) are immobilized at the capture zone due to binding to the indicator molecules (9) at the cleavage site. Due to the relative excess of labelled binding molecule (7) to binding sites at the capture zone some labelled binding molecules (7) still flow through to the control zone and beyond. Further labelled molecules (11) also pass through the device to the control zone where they are immobilized by binding to the immobilized further binding molecules (6). Thus, presence of enzyme cleavage activity is displayed as a signal both at the capture zone and the control zone. Excess sample, potentially containing cleavage products of the indicator molecule that do not contain the biotin capture site (10), flows into the absorbent pad (8).

(40) It should be noted that the control zone is optional. The presence or absence of enzyme cleavage activity in the sample can be monitored solely based upon the presence or absence of a corresponding signal at the capture zone.

(41) FIG. 3 shows the visual read-out of the assay (shown in FIG. 2) as levels of MMP activity in the test sample are increased. As can readily be seen, the signal at the control zone (1) is constant as MMP amounts increase. In contrast, as MMP amounts increase, the signal at the capture zone (2) also increases. This is due to cleavage of the indicator molecule at the cleavage site by MMP activity. This reveals a binding site, enabling binding of the binding molecules which is detected at the capture zone (2) via interaction between capture molecules defining the capture zone and the capture site of the indicator molecules.

(42) FIG. 4 is a schematic view of one specific enzyme detection device in accordance with the present invention. The table below provides a legend for the figure and specifies the exact longitudinal dimensions and position of each of the card components in this particular embodiment. Of course, the dimensions and positions may be varied as would be readily understood by one skilled in the art.

(43) TABLE-US-00003 Component Size Position from Datum point Backing card (1) 60 mm 0 mm Nitrocellulose Membrane (2) 25 mm 20 mm Conjugate Pad (3) 17 mm 5 mm Sample Pad (4) 10 mm 0 mm Absorbent Pad (5) 22 mm 38 mm

(44) FIG. 5 shows an example of synthesis of a structurally constrained indicator molecule. It should be noted that additional spacer or linker regions may be included between the cleavage region and the site of attachment of the scaffold molecule.

(45) In FIG. 5A initially, a linear peptide (1) is synthesised, for example using solid phase Fmoc chemistry. The peptide may be purified for example by High Performance Liquid Chromatography (HPLC). The peptide is then constrained, or cyclised, by reaction between thiol groups on the peptide (2) and the scaffold molecule (3). This reaction produces a structurally constrained clipped peptide (4).

(46) In FIG. 5B, the indicator molecule is synthesised to include the capture site (1), for example by synthesis of the linear peptide on a pre-loaded Biotin-PEG resin.

(47) FIG. 6 shows schematically the ability of the binding molecules used in the invention to bind exclusively to the cleaved indicator molecule. In the absence of enzyme cleavage activity, the structurally constrained indicator molecule (1) is not bound by the antibody binding molecule (2). This antibody is generated using the cleaved indicator molecule (3) as antigen and thus only binds to this open form of the molecule.

(48) FIGS. 13 and 14 show a range of suitable scaffold molecules for use in the invention.

(49) FIG. 15 shows, in schematic form, some attachment options for scaffold molecules to the indicator molecules. FIG. 15A shows products of cleavage at a single cleavage site and FIG. 15B shows products of cleavage at two separate cleavage sites.

(50) A Human Neutrophil Elastase substrate is shown in FIG. 23 in the form of an indicator molecule of the invention. Synthesis of this substrate/indicator molecule is discussed in Examples 9 and 10 below. The substrate contains the amino acid sequence CQESIRLPGC (SEQ ID NO: 3) which is cyclised using an appropriate scaffold (in this case 1,3-dibromomethylbenzene). Cyclisation utilises the thiol groups provided by the two cysteine residues to link with the scaffold. Cleavage by HNE at the peptide bond between the isoleucine and arginine residues opens up the structure to reveal a new binding site (or cryptotope). HNE cleaves the substrate at a single site to produce a reliable binding site. The additional tyrosine residue (see SEQ ID NO: 4) facilitates attachment to further moieties such as a carrier protein as discussed herein.

(51) The invention will be further understood with reference to the following experimental examples.

EXAMPLES

(52) Throughout the examples and figures the invention may be referred to as Ultimate ELTABA

Example 1

A Lateral Flow Platform of the Invention for Detection of Matrix Metalloprotease-9 (MMP-9)

(53) A kit comprises the following components:

(54) 1) A device for sample collection (e.g. for urine)

(55) 2) A lateral flow test-strip, which is mounted in a plastic case. The test strip has a capture zone comprising polystreptavidin as a first test line across the flow-path of the test strip. A second capture zone comprising anti-chicken antibodies adsorbed as a control line across the flow-path of the test strip, downstream of the test line may be included as a control line. There is an observation window in the plastic case through which to view the test and control line. There is also an integrated sample-receiving pad, upstream of the first test line. In addition, the test strip has gold particles bearing sheep antibody (CF1522) dried into the test strip, downstream of the sample-receiving pad which can be reconstituted by the addition of the sample.
3) A tube, in which the sample collection device may be placed, together with the indicating molecule.
4) An indicator molecule containing the cleavable sequence, in this example, (GPQGIFGQ) which carries a terminal biotin group connected via a polyethylene glycol spacer/linker which allows it to form a complex with the capture line, polystreptavidin.
The Test Strip

(56) A test strip for the detection of protease activity in a sample was constructed in accordance with the present invention, as described below. The assay was based on the cleavage of the indicator molecule in the presence of various MMP's to expose an epitope visible to the Sheep antibody (CF1522) conjugated to gold particles.

(57) The methods used were all in accordance with standard procedures well known in the art.

(58) A. Preparation of CF1522:40 nm Gold Conjugate

(59) Affinity purified sheep antibody CF1522 (Ig Innovations, CF1522) was conjugated to 40 nm gold particles at a concentration giving an OD of 5 at 520 nm (BBI International, GC40). The antibody was loaded at a concentration of 15 g/ml in a 20 mM BES buffer pH 7.8. 0.2% BSA (Sigma, A7906) was used as a blocking solution to minimise non-specific binding.

(60) B. Preparation of Gold-Impregnated Conjugate Pads

(61) A glass fibre conjugate pad (Millipore, G041, 17 mm300 mm) was sprayed with CF1522:40 nm gold conjugate (Mologic) at OD4, diluted in gold drying buffer (1 M Tris, 150 mM sodium chloride, 20 mM sodium Azide, 3% BSA, 5% Sucrose, 1% Tween 20 at pH 9.4) at 0.8 l/mm with the Isoflow dispenser (15 mm spray height). The processed conjugate band was dried in a tunnel dryer at 60 C. at a speed of 5 mm/sec. The dried gold conjugate-impregnated conjugate pads were stored dried in a sealed foil pouch with desiccant at room temperature.

(62) C. Preparation of Antibody-Impregnated Nitrocellulose Membrane

(63) All reagents were striped on Unistart CN140 membrane (Sartorius, CN140, 25 mm300 mm) at a dispense rate of 0.1 l/mm. A test line polystreptavidin (BBI, Polystrep N 01041048K) at a concentration of 1 mg/ml was positioned 7 mm from base of membrane. Processed membrane was dried in a tunnel dryer at 60 C. at a speed of 10 mm/sec. The dried antibody-impregnated Nitrocellulose Membrane was stored in a sealed foil pouch with desiccant at room temperature.

(64) D. Card Assembly

(65) A test card was assembled according to the following procedure and in accordance with FIG. 4 which specifies the exact longitudinal dimensions and position of each of the card components. 1. A 60300 mm piece of clear plastic film with a release liner protected adhesive, serving as the back laminate, designated 1 in FIG. 4, (G&L Precision Die Cutting, GL-48077) was placed on top of a worktable. The release liner was peeled to expose the adhesive. 2. The reaction membrane (prepared as in section C) was attached on top of the adhesive side of the back cover, 20 mm from the lower end. 3. The impregnated conjugate pad (prepared as in section B) was attached on top of the back cover with 2 mm overlap on top of the reaction membrane. 4. The sample pad (MDI, FR-1, 10300 mm) was placed on top of the back cover with 5 mm overlap on top of the conjugate pad. 5. The absorbent pad (Gel blotting paper, Ahlstrom, grade 222, 22300 mm) was placed on top of the upper side of the back cover with a 2 mm overlap on top of the reaction membrane.

(66) The card was trimmed to 5 mm width strips using an automated die cutter (Kinematic, 2360) and assembled into plastic housings (Forsite). The devices were closed using a pneumatic device clamp specifically manufactured for these devices at Mologic.

(67) The table lists the strip components and respective positioning on a backing card.

(68) TABLE-US-00004 Component Size Position from Datum point Backing card (1) 60 mm 0 mm Nitrocellulose Membrane (2) 25 mm 20 mm Conjugate Pad (3) 17 mm 5 mm Sample Pad (4) 10 mm 0 mm Absorbent Pad (5) 22 mm 38 mm

(69) Buffer standards were produced containing different concentrations of active MMP-9 (Alere San Diego) ranging from 1000 ng/ml down to 1 ng/ml.

(70) STEP 1: Each standard was placed in a collection device with a defined amount of peptide (25 ng/test). The collection device was rotated vigorously in order for the sample to mix sufficiently with the substrate solution. This reaction mixture was incubated at ambient temperature for a defined period of time (e.g. 10 minutes).

(71) STEP 2: At the end of the incubation period, a defined volume of liquid was dropped onto the sample receiving pad which subsequently made contact with the conjugate pad and re-hydrated the dried CF1522 antibody attached to the gold particles. Intact indicator molecule was not recognised by the gold conjugate and migrated in an uncomplexed state towards the polystreptavidin test line where it was immobilised via the biotin attached to the indicator molecule. Any MMP-9 present in the sample cleaved the indicator molecule at the cleavage site, exposing the recognisable epitope thus allowing the gold conjugate to form a complex with the cleaved stub.

(72) The lines that were formed were assessed by their relative intensities. The presence of a test line indicated that there was protease present in the test sample. A negative test line indicated a zero or low level of protease that was below the detectable limit. Stages in between these extremes indicated different levels of protease in the test sample. The intensity of the developed coloured lines was measured visually and with a Forsite Lateral flow device reader. A semi-quantitative scoring system with a scale of 0-10, in which 1 was the lowest detectable colour intensity and 10 was the highest observed colour intensity was used for the visual readings.

(73) FIG. 7 (FIGS. 7A and 7B) demonstrates the sensitivity of the assay when run with spiked MMP-9 buffer samples. The detectable limit for MMP-9 was approximately 4 ng/ml with a sample volume of 75 l. FIG. 7A shows reader values across the entire concentration range of MMP-9, whereas FIG. 7B is an expanded view at MMP-9 concentrations between 0 and 15 ng/ml.

(74) The reader units are displayed in the table below where a value above 400 was deemed a positive result:

(75) TABLE-US-00005 ng/ml MMP9 reader value 1000 6770 500 6729 250 6225 125 5581 62.5 3826 31.25 2029 15.625 882 7.8125 524 3.90625 413 1.953125 343 0.9765625 338 0 312

Example 2

Matrix Metalloprotease (MMP) Specificity of a Lateral Flow Format of the Invention

(76) The kit and test strip synthesis were performed as for Example 1.

(77) Various MMP's (Enzo) were prepared in buffer (Aq. Solution of 50 mM Tris, 150 mM sodium chloride, 20 mM sodium azide, 1% vol/vol Tween 20, at pH 8.0) at 0.5 g/ml.

(78) STEP 1: Each MMP solution was placed in a collection device with a defined amount of peptide (25 ng/test). The collection device was rotated vigorously in order for the sample to mix sufficiently with the substrate solution. This reaction mixture was incubated at ambient temperature for a defined period of time (e.g. 10 minutes).

(79) STEP 2: At the end of the incubation period, a defined volume of liquid was dropped onto the sample receiving pad which subsequently made contact with the conjugate pad and re-hydrated the dried CF1522 antibody attached to the gold particles. Intact indicator molecule was not recognised by the gold conjugate and migrated in an uncomplexed state towards the polystreptavidin test line where it was immobilised via the biotin attached to the indicator molecule. Any MMP-9 present in the sample cleaved the indicator molecule at the cleavage site, exposing the recognisable epitope thus allowing the gold conjugate to form a complex with the cleaved stub.

(80) The lines that were formed were assessed by their relative intensities. The presence of a test line indicated that there was protease present in the test sample. A negative test line indicated a zero or low level of protease that was below the detectable limit. Stages in between these extremes indicated different levels of protease in the test sample. The intensity of the developed coloured lines was measured visually and with a Forsite Lateral flow device reader. A semi-quantitative scoring system with a scale of 0-10, in which 1 was the lowest detectable colour intensity and 10 was the highest observed colour intensity was used for the visual readings.

(81) FIG. 8 demonstrates that this version of the invention uses a cleavable sequence that is biased towards MMP13, MMP12, MMP9, MMP8 and MMP2. Other versions of this invention may use sequences with different targets depending on the application required.

(82) The table below shows the read-out values for each of the MMPs tested:

(83) TABLE-US-00006 MMP Reader value 1 477.5 2 1608.5 3 336.5 7 373 8 1140.5 9 3844 10 444 11 279.5 12 1252.5 13 6348.5

Example 3

Detection of Enzyme Activity in Urine

(84) The kit and test strip synthesis were performed as for Example 1.

(85) Samples were collected from healthy volunteers (9) and from patients suffering from a respiratory disease. Samples were donated from nine patients with Cystic Fibrosis (CF) and seven patients with Chronic Obstructive Pulmonary Disease (COPD) and stored at 80 C. until used.

(86) STEP 1: Each sample was placed in a collection device with a defined amount of peptide (25 ng/test). The collection device was rotated vigorously in order for the sample to mix sufficiently with the substrate solution. This reaction mixture was incubated at ambient temperature for a defined period of time (e.g. 10 minutes).

(87) STEP 2: At the end of the incubation period, a defined volume of liquid was dropped onto the sample receiving pad which subsequently made contact with the conjugate pad and re-hydrated the dried CF1522 antibody attached to the gold particles. Intact indicator molecule was not recognised by the gold conjugate and migrated in an uncomplexed state towards the polystreptavidin test line where it was immobilised via the biotin attached to the indicator molecule. Any MMP-9 present in the sample cleaved the indicator molecule at the cleavage site, exposing the recognisable epitope thus allowing the gold conjugate to form a complex with the cleaved stub.

(88) The lines that were formed were assessed by their relative intensities. The presence of a test line indicated that there was protease present in the test sample. A negative test line indicated a zero or low level of protease that was below the detectable limit. Stages in between these extremes indicated different levels of protease in the test sample. The intensity of the developed coloured lines was measured visually and with a Forsite Lateral flow device reader. A semi-quantitative scoring system with a scale of 0-10, in which 1 was the lowest detectable colour intensity and 10 was the highest observed colour intensity was used for the visual readings.

(89) FIG. 9 demonstrates that measurable amounts of active proteases (in particular MMPs, including MMP-9) can be found in urine samples and that higher levels are present in samples obtained from patients with a respiratory disease. A significant difference was observed with COPD samples when compared to samples collected from healthy controls (P=0.03) and CF samples to healthy controls (P=0.02).

Example 4

Detection of Enzyme Activity in Wound Fluid

(90) The kit and test strip synthesis were performed as for Example 1.

(91) Wound samples from 18 patients were tested on the ultimate ELTABA device to measure active MMP's in this biologic matrix. The samples were extracted from a swab (Copan, 552C.US) in MMP buffer buffer (Aq. Solution of 50 mM Tris, 100 mM sodium chloride, 10 mM Calcium Chloride, 50 M 20 mM zinc chloride, 0.025% Brij 35, 0.05% sodium azide at pH 8.0) and then frozen at 20 C. until use. The addition d a chelating agent (5 mM EDTA) determined the specificity of the device to calcium dependent enzymes e.g. MMP's.

(92) STEP 1: Each wound sample was diluted 1 in 20 in MMP buffer and 75 l was placed in a collection device with a defined amount of peptide (25 ng/test). The collection device was rotated vigorously in order for the sample to mix sufficiently with the substrate solution. This reaction mixture was incubated at ambient temperature for a defined period of time (e.g. 10 minutes).

(93) STEP 2: At the end of the incubation period, a defined volume of liquid was dropped onto the sample receiving pad which subsequently made contact with the conjugate pad and re-hydrated the dried biotin attached to the gold particles. Intact indicator molecule was not recognised by the gold conjugate and migrated in an uncomplexed state towards the Polystreptavidin test line where it was immobilised via the biotin attached to the indicator molecule. Any MMP-9 present in the sample cleaved the indicator molecule at the cleavage site, exposing the recognisable epitope thus allowing the gold conjugate to form a complex with the cleaved stub.

(94) The lines that were formed were assessed by their relative intensities. The presence of a test line indicated that there was protease present in the test sample. A negative test line indicated a zero or low level of protease that was below the detectable limit. Stages in between these extremes indicated different levels of protease in the test sample. The intensity of the developed coloured lines was measured visually and with a Forsite Lateral flow device reader. A semi-quantitative scoring system with a scale of 0-10, in which 1 was the lowest detectable colour intensity and 10 was the highest observed colour intensity was used for the visual readings.

(95) FIG. 10 shows that addition of EDTA to the wound samples inhibits the readout, confirming the presence of MMP in the samples and also confirms that the assay is specifically measuring active MMPs.

Example 5

Comparison of Sensitivity of the Invention to a Commercial MMP-9 Activity Assay Kit

(96) The commercial kit is designed for specifically detecting MMP-9 in biologic samples such as culture medium, serum, plasma, synovial fluid, and tissue homogenate. A monoclonal anti-human MMP is used to pull down both pro and active forms of MMP from the mixture first, and then the activity of MMP9 is quantified using fluorescence resonance energy transfer (FRET) peptide. An MMP-9 standard AMPA activated in-house was run on both the kit and a lateral flow format of the invention at a range of 250 ng/ml-4 ng/ml. For the commercial assay the MMP-9 was diluted in an MMP buffer supplied in the kit and a Tris buffer saline 1% Tween20 for lateral flow devices.

(97) The lateral flow kit and test strip synthesis were performed as for Example 1.

(98) Buffer standards were produced containing different concentrations of active MMP-9 (Alere San Diego) ranging from 250 ng/ml down to 4 ng/ml in a Tris buffer saline 1% Tween (Aq. Solution of 50 mM Tris, 150 mM sodium chloride, 20 mM sodium azide, 1% vol/vol Tween 20, at pH 8.0).

(99) STEP 1: Each standard was placed in a collection device with a defined amount of peptide (25 ng/test). The collection device was rotated vigorously in order for the sample to mix sufficiently with the substrate solution. This reaction mixture was incubated at ambient temperature for a defined period of time (e.g. 10 minutes).

(100) STEP 2: At the end of the incubation period, a defined volume of liquid was dropped onto the sample receiving pad which subsequently made contact with the conjugate pad and re-hydrated the dried CF1522 antibody attached to the gold particles. Intact indicator molecule was not recognised by the gold conjugate and migrated in an uncomplexed state towards the polystreptavidin test line where it was immobilised via the biotin attached to the indicator molecule. Any MMP-9 present in the sample cleaved the indicator molecule at the cleavage site, exposing the recognisable epitope thus allowing the gold conjugate to form a complex with the cleaved stub.

(101) The lines that were formed were assessed by their relative intensities. The presence of a test line indicated that there was protease present in the test sample. A negative test line indicated a zero or low level of protease that was below the detectable limit. Stages in between these extremes indicated different levels of protease in the test sample. The intensity of the developed coloured lines was measured visually and with a Forsite Lateral flow device reader. A semi-quantitative scoring system with a scale of 0-10, in which 1 was the lowest detectable colour intensity and 10 was the highest observed colour intensity was used for the visual readings.

(102) FIG. 11 (FIGS. 11A and 11B) is a graph comparing the ability of a commercially available active MMP-9 assay kit and the assay of the invention to detect MMP9. FIG. 11A shows reader values across the entire concentration range of MMP-9, whereas FIG. 11B is an expanded view at MMP-9 concentrations between 0 and 50 ng/ml. Both figures demonstrate that the assay of the invention produced a steeper curve. According to both assays, colour development as shown by the absorbance values was seen at 4 ng/ml MMP9, the lowest standard tested.

(103) Numerical read-outs for each assay are shown in the table below:

(104) TABLE-US-00007 ng/ml MMP9 Reference assay Ultimate ELTABA 250 204466.5 6225 125 162622 5581 62.5 112706.5 3826 31.25 62301.5 2029 15.625 31295 882 7.8125 13140.5 524 3.90625 7601 413 0 3818.5 312

Example 6

Testing of Substrate in Both ELISA and LF Format

(105) ELISA Format

(106) 1) A device for sample collection (e.g. for urine)

(107) 2) A 96 well plate coated with polystreptavidin

(108) 3) A tube, in which the sample collection device may be placed, together with the indicating molecule.

(109) 4) An indicator molecule containing the cleavable sequence, in this example, (GPQGIFGQ) which carries a terminal biotin group connected via a polyethylene glycol spacer/linker which allows it to form a complex with the capture line, polystreptavidin.

(110) 5) A sheep antibody CF1522 conjugated to alkaline phosphatase (AP)

(111) 6) An Alkaline phosphatase substrate p-nitrophenylphosphate (pNPP) that enables the development of a soluble yellow reaction product that may be read at 405 nm.

(112) Samples were collected from healthy volunteers (9) and from patients suffering from a respiratory disease. Samples were donated from nine patients with Cystic Fibrosis (CF) and seven patients with Chronic Obstructive Pulmonary Disease (COPD) and stored at 80 C. until used.

(113) STEP 1: Each sample was placed in a collection device with a defined amount of peptide (25 ng/test). The collection device was rotated vigorously in order for the sample to mix sufficiently with the substrate solution. This reaction mixture was incubated at ambient temperature for a defined period of time (e.g. 10 minutes).

(114) STEP 2: At the end of the incubation period, a defined volume of sample was added to the streptavidin plate (Nunc, 442404) and incubated for a further 1 hr at ambient where the biotin labelled indicator molecule becomes immobilized by the streptavidin bound to the plate.

(115) STEP 3: The plate was washed 3 times with 100 l in a wash buffer, Tris buffer saline 0.1% Tween (Aq. Solution of 50 mM Tris, 150 mM sodium chloride, 20 mM sodium azide, 0.1% vol/vol Tween 20, at pH 8.0).

(116) STEP 4: CF1522-AP (Mologic) was diluted 1/500 in 1% BSA in PBST and incubated on the plate for 1 hr at ambient. The antibody will form a complex with the cleaved stubs exposed by any MMP present in the sample and in the absence of the cleaved stub there will be no binding of the antibody.

(117) STEP 5: The plate was washed 3 times with 100 l in a wash buffer, Tris buffer saline 0.1% Tween (Aq. Solution of 50 mM Tris, 150 mM sodium chloride, 20 mM sodium azide, 0.1% vol/vol Tween 20, at pH 8.0).

(118) STEP 6: The plate was incubated with pNPP substrate and then read at 405 nm after 30 minute incubation at 37 C. An MMP9 standard curve is represented in FIG. 14b used as a reference. The colour of the wells indicate different levels of protease in the test sample represented by the OD 405 nm in FIG. 14b,

(119) Lateral Flow Format

(120) The kit and test strip synthesis were performed as for Example 1.

(121) Buffer standards were produced containing different concentrations of active MMP-9 (Alere San Diego) ranging from 50 ng/ml down to 0.39 ng/ml and 62.5 ng/ml down to 0.97 ng/ml for the ELISA and lateral flow device respectively.

(122) STEP 1: Each sample was placed in a collection device with a defined amount of peptide (25 ng/test). The collection device was rotated vigorously in order for the sample to mix sufficiently with the substrate solution. This reaction mixture was incubated at ambient temperature for a defined period of time (e.g. 10 minutes).

(123) STEP 2: At the end of the incubation period, a defined volume of liquid was dropped onto the sample receiving pad which subsequently made contact with the conjugate pad and re-hydrated the dried CF1522 antibody attached to the gold particles. Intact indicator molecule was not recognised by the gold conjugate and migrated in an uncomplexed state towards the polystreptavidin test line where it was immobilised via the biotin attached to the indicator molecule. Any MMP-9 present in the sample cleaved the indicator molecule at the cleavage site, exposing the recognisable epitope thus allowing the gold conjugate to form a complex with the cleaved stub.

(124) The lines that were formed were assessed by their relative intensities. The presence of a test line indicated that there was protease present in the test sample. A negative test line indicated a zero or low level of protease that was below the detectable limit. Stages in between these extremes indicated different levels of protease in the test sample. The intensity of the developed coloured lines was measured visually and with an NES Lateral flow device reader.

(125) The results of an MMP9 standard curve can be seen in FIG. 12. FIG. 12 demonstrates that the two MMP9 standard curves produced by the ELISA and the Lateral Flow are similar with sensitivity down to 4 ng/ml.

(126) The numerical read-outs from the two assays are also shown in the table below:

(127) TABLE-US-00008 ELISA Lateral Flow standard curve standard curve ng/ml ng/ml Reader MMP9 OD405 MMP9 value 50.00 0.50 62.50 3826.00 25.00 0.27 31.25 2029.00 12.50 0.18 15.63 882.00 6.25 0.17 7.81 524.00 3.13 0.13 3.91 413.00 1.56 0.14 1.95 343.00 0.78 0.14 0.98 338.00 0.39 0.14 0.00 312.00

Example 7

Synthesis of an Example Indicator Molecule

(128) A peptide termed MOL386 (amino acid sequence: CGPQGIFGQC) was synthesised on solid phase using Fmoc-chemistry. Briefly, synthesis was performed on a microwave assisted automated synthesiser (CEM Liberty). Coupling steps were carried out on PEG-polystyrene resin preloaded with Fmoc-Cys(Trt) in DMF solvent with a fivefold excess of amino acid building block, HBTU activator and a tenfold excess of DIPEA base. Deprotection steps were carried out in 5% Piperazine/DMF. Completed peptide resin was dried and then cleaved using 95% TFA, 2.5% TIPS and 2.5% water for 2 hours. TFA liquors were dried in vacuo and precipitated in ether to afford colourless peptide solid. Recovered peptide was freeze dried from 50% acetonitrile and purified by HPLC (FIG. 16) using a C18 reverse phase column and a gradient of 5% acetonitrile/water (0.1% TFA) to 100% acetonitrile (0.1% TFA). Isolated fractions were combined and freeze dried and analysed by electrospray mass spectrometry (FIG. 17) to identify target peptide (expected MH.sub.+ 1010.17, measured 1010.3). The biotinylated form (CGPQGIFGQC-PEG-biotin) was synthesised from preloaded Biotin-PEG-NovaTag Resin (Merck) (expected MH.sub.+ 1438.76, measured 1439.7, FIG. 18). The biotin provides a capture site for immobilization of the indicator molecule.

(129) Attachment of the Scaffold Molecule (Synthesis of Cyclised Peptide)

(130) Peptide (1 mg) was dissolved in PBS 250 ul along with 1 mg of 1,3-dibromomethylbenzene and agitated gently overnight. The reaction was then diluted with 1 ml of water and injected directly on to HPLC for purification using a C18 reverse phase column and a gradient of 5% acetonitrile/water (0.1% TFA) to 100% acetonitrile (0.1% TFA). Product peak was isolated and freeze dried to afford a colourless solid (expected MH.sub.+ 1112.30, measured 1112.8, FIG. 19). The same procedure was used for the biotinylated peptide (expected MH.sub.+ 1540.89, measured 1539.8, FIG. 20).

Example 8

Test Format Generation

(131) Antibodies were generated to recognise a cleaved peptide sequence. In this example (GPQGIFGQ), a target for MMP digestion, is used in an immunoassay to measure the enzyme activity in a clinical sample. The antibodies were raised to peptide KLH conjugates using methods known to those skilled in the art. Sheep antibodies CF1522 and CF1523 were generated to recognise cleaved stub IFGQ whereas sheep antibodies CF1524 and CF1525 were generated to recognise cleaved stub GPQG. The antibodies were affinity purified using the specific peptides they were raised against and then analysed by ELISA to determine the most appropriate assay format to give the best sensitivity.

(132) Peptides containing the cleavable sequence (GPQGIFGQ) were synthesised with a biotin or Pegylated biotin attached to either the C-terminus (MOL038 and PCL008-A2 respectively) or the N-terminus (MOL310 and MOL378 respectively).

(133) TABLE-US-00009 Peptide Sequence MOL038 Biotin-GPQGIFGQESIRLPGCPRGVNPVVS PCL008-A2 Biotin-PEG-Asp-AEEAc-AEEAc-GPQGI FGQESIRLPGCPRGVNPVVS MOL310 SIRLPGCPRGVNPVVSGPQGIFGQ-Biotin MOL378 SIRLPGCPRGVNPVVSGPQGIFGQ-AEEAc- AEEAc-PEG-AspBiotin

(134) The peptide can be anchored to either streptavidin capture via the biotin or to sheep antibody CF1060 capture via the ALP sequence. The proposed formats shown schematically in FIG. 1 were evaluated.

(135) ELISA Format

(136) 1) A device for sample collection (e.g. for urine)

(137) 2) A 96 well plate coated with polystreptavidin (Nunc, 442404) or CF1060 overnight at ambient (Nunc, Maxisorb)

(138) 3) A tube, in which the sample collection device may be placed, together with the indicating molecule.

(139) 4) An indicator molecule containing the cleavable sequence, in this example, (GPQGIFGQ) which carries a terminal biotin group which may be connected via a polyethylene glycol spacer/linker on the N or the C-terminus.

(140) 5) Sheep antibodies CF1522, CF1523, CF1524 and CF1525 conjugated to alkaline phosphatase (AP)

(141) 6) An Alkaline phosphatase substrate p-nitrophenylphosphate (pNPP) that enables the development of a soluble yellow reaction product that may be read at 405 nm.

(142) Active MMP9 (Alere San Diego) was diluted to 2, 0.25, 0.062, 0.0156 and 0.039 g/ml in MMP buffer (Aq. Solution of 50 mM Tris, 100 mM sodium chloride, 10 mM Calcium Chloride, 50 M 20 mM zinc chloride, 0.025% Brij 35, 0.05% sodium azide at pH 8.0)

(143) STEP 1: Each MMP9 standard was placed in a collection device with a defined amount of each peptide (20 ng/test). The collection device was rotated vigorously in order for the sample to mix sufficiently with the substrate solution. This reaction mixture was incubated at ambient temperature for a defined period of time (e.g. 30 minutes).

(144) STEP 2: At the end of the incubation period, a defined volume of sample was added to the streptavidin plate and CF1060 sensitised plate and incubated for a further 1 hr at ambient where the peptides becomes immobilized by the streptavidin or CF1060 bound to the plate.

(145) STEP 3: The plate was washed 3 times with 100 l in a wash buffer, Tris buffer saline 0.1% Tween (Aq. Solution of 50 mM Tris, 150 mM sodium chloride, 20 mM sodium azide, 0.1% vol/vol Tween 20, at pH 8.0).

(146) STEP 4: sheep antibodies conjugated to Alkaline Phosphatase (Mologic) were diluted 1/500 in 1% BSA in PBST and incubated on the plate for 1 hr at ambient. The antibody will form a complex with the cleaved stubs exposed by any MMP9 present in the sample, in the absence of the cleaved stub there will be no binding of the antibody.

(147) STEP 5: The plate was washed 3 times with 100 l in a wash buffer, Tris buffer saline 0.1% Tween (Aq. Solution of 50 mM Tris, 150 mM sodium chloride, 20 mM sodium azide, 0.1% vol/vol Tween 20, at pH 8.0).

(148) STEP 6: The plate was incubated with pNPP substrate and then read at 405 nm after 30 minute incubation at 37 C. MMP9 standard curves are represented in FIG. 21 for all combinations. A difference in colour of the wells indicates different levels of protease in the test sample represented by the OD 405 nm.

(149) FIG. 21 shows the results of testing each format. With a streptavidin capture line, the selected peptide is MOL378 with sheep antibody CF1522 and PCL008-A2 with sheep antibody CF1525 as predicted. Both peptides contained a PEG-Asp-AEEAc-AEEAc required to reduce any steric hindrance. With a CF1060 capture line, the selected peptide is MOL038 or PCL008-A2 with sheep antibody CF1522 and MOL378 with sheep antibody CF1525 as predicted. The performance of the best combinations is shown in FIG. 22. Here, format 4 using sheep antibody CF1522 with peptide MOL378 shows the most promise.

Example 9

Synthesis of a Human Neutrophil Elastase Sensitive Indicator Molecule

(150) A peptide termed MOL 488 (amino acid sequence YCQESIRLPGCSEQ ID NO: 4) was synthesised on solid phase using Fmoc-chemistry. Briefly, synthesis was performed on a microwave assisted automated synthesiser (CEM Liberty). Coupling steps were carried out on PEG polystyrene resin with a fivefold excess of amino acid building block, DIC and Oxyma. Deprotection steps were carried out in 20% Piperidine/DMF. Completed peptide resin was dried and then cleaved using 95% TFA, 2.5% TIPS and 2.5% water for 2 hours. TFA liquors were dried in vacuo and precipitated in ether to afford colourless peptide solid. Recovered peptide was freeze dried from 50% acetonitrile and purified by HPLC using a C18 reverse phase column and a gradient of 5% acetonitrile/water (0.1% TFA) to 100% acetonitrile (0.1% TFA). Isolated fractions were combined and freeze dried and analysed by electrospray mass spectrometry (expected MH.sub.+ 1268.5, measured 1267.861.39)see FIG. 28

(151) Attachment of the Scaffold Molecule (Synthesis of Cyclised Peptide)

(152) Peptide (1 mg) was dissolved in PBS 250 ul along with 1 mg of 1,3-dibromomethylbenzene and agitated gently overnight. The reaction was then diluted with 1 ml of water and injected directly on to HPLC for purification using a C18 reverse phase column and a gradient of 5% acetonitrile/water (0.1% TFA) to 100% acetonitrile (0.1% TFA). Product peak was isolated and freeze dried to afford a colourless solid (expected MH.sub.+ 1370.6, measured 1371.93.8); see FIG. 29.

(153) Preparative Enzymatic Cleavage of Cyclised MOL488

(154) Cyclised Peptide (1 mg) was dissolved in Cleavage Buffer (100 nm Tris pH 8.0. 0.05% Brij) at a final concentration of 5 mg/ml. Enzyme Human Neutrophil elastase (Leeblo Solutions inc.) was added to a final concentration of U/ml. To follow reaction progress timed aliquots were quenched in 5 volumes of starting buffer (5% acetonitrile, 0.1% TFA) and checked on HPLC. A new product peak evolved over time and after approximately three hours the reaction was stopped and the product fraction purified by HPLC (expected MH.sub.+ 1388.6, measured 1388.82.5)see FIG. 30.

(155) The same procedure was also followed in respect of a similar substrate but in this case lacking the tyrosine residue (SEQ ID NO: 3).

(156) HPLC results are summarised in FIG. 24. It can be seen from FIG. 24A that a single cleavage product results from HNE activity on the peptide. FIG. 24B presents a time course showing the relative increase in product and decrease in substrate over time.

(157) FIG. 25 presents mass spectrometric data confirming that the substrate is cleaved at a single site and that the substrate otherwise remains intact. FIG. 25A is the substrate plot and FIG. 25B is the hydrolysed product.

Example 10

Conjugation Methods

(158) To conjugate the protease-digested peptide product to a carrier protein to immunise and develop antibodies, a chemistry orthogonal to the Clip thiol alkylation route needs to be applied. A combination of three different chemistries (diazo, oxime and triazole) are considered in this instance to achieve conjugation. In the first option (FIG. 26) the peptide can be synthesised with a pendant tyrosine residue. The heterobifunctional reagent FBDP (Sigma) is used to conjugate an aminooxy linker (Berry Associates) on to the phenol group of the tyrosine creating a pendant azide tail. This in turn can be conjugated to a carrier protein labelled with an alkyne reagent. Alternatively, the peptide can be synthesised with an aminooxy terminus (FIG. 27) and this can then be crosslinked directly to tyrosine residues on the carrier protein using the FBDP reagent.

(159) The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken from other aspects of the invention (including in isolation) as appropriate. Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.