AU2003210267A1 - Diagnostic method for stroke - Google Patents

Description

WO 03/069346 PCT/EPO3/01462 - 1 DIAGNOSTIC METHOD FOR STROKE BACKGROUND OF THE INVENTION 5 Field of the invention This invention relates to a diagnostic method for stroke. Description of the related art 10 Stroke has the third highest death-rate in industrial countries. It is caused either by bleeding in the brain from a ruptured blood vessel (haemorrhagic stroke) or by obstruction of a blood vessel in the brain (ischaemic or thrombotic stroke). Stroke results from either a permanent or a transient reduction in cerebral blood flow. This 15 reduction in flow is, in most cases, caused by the arterial occlusion due to either an embolus or a local thrombosis. Depending on the localisation of brain injury and the intensity of necrosed neurones, stroke symptoms can become a life handicap for patients and the death rate from stroke events approaches 30%. 20 Recently, S 100OB was described as a potential biochemical marker for stroke diagnosis, see U.Missler et al., "S 100 protein and neuron-specific enolase concentrations in blood as indicators of infarct volume and prognosis in acute ischemia stroke", Stroke 1997; 28:1956-60. However, S 100B has also been reported as a useful marker for early detection of metastases of melanoma and cerebral 25 complications from head injury and cardiac surgery. Thus, the sensitivity and specificity of the S 100B test were limited to 44% and 67%, respectively, see M.Takahashi et aL, "Rapid and sensitive immunoassay for the measurement of serum S100B using isoform-specific monoclonal antibody", Clin. Chem. 1999; 45:1307-11. Development of new stroke markers would help clinicians to establish early diagnosis. 30 WO 03/069346 PCT/EPO3/01462 2 WO 01/42793 relates to a diagnostic assay for stroke in which the concentration of heart or brain fatty acid binding protein (H-FABP or B-FABP) is determined in a sample of body fluid. 5 US-A-6225047 describes the use of retentate chromatography to generate difference maps, and in particular a method of identifying analytes that are differentially present between two samples. One specific method described therein is laser desorption mass spectrometry. 10 WO 01/25791 describes a method for aiding a prostate cancer diagnosis, which comprises determining a test amount of a polypeptide marker, which is differentially present in samples of a prostate cancer patient and a subject who does not have prostate cancer. The marker may be determined using mass spectrometry, and preferably laser desorption mass spectrometry. 15 Development of new non-invasive stroke markers for body fluids and new methods of determining the markers would help clinicians to establish early diagnosis. This problem has now been solved by the present invention. 20 SUMMARY OF THE INVENTION The present invention provides a method of diagnosis of stroke or the possibility thereof in a subject suspected of suffering from stroke, which comprises subjecting a sample of body fluid taken from the subject to mass spectrometry, thereby to 25 determine a test amount of a polypeptide in the sample, wherein the polypeptide is differentially contained in the body fluid of stroke-affected subjects and non-stroke affected subjects, and has a molecular weight in the range of from 3000 to 30000; and determining whether the test amount is consistent with a diagnosis of stroke. The test amount can also be used to determine the type of stroke that is diagnosed, in particular 30 whether it is of the ischaemic or haemorrhagic type.

WO 03/069346 PCT/EPO3/01462 -3 The invention also provides use of a polypeptide which is differentially contained in a body fluid of stroke-affected subjects and non-stroke-affected subjects, the polypeptide having a molecular weight in the range of from 3000 to 30000 and being 5 determinable by mass spectrometry, for diagnostic, prognostic and therapeutic applications. The invention further provides a kit for use in diagnosis of stroke, comprising a probe for receiving a sample of body fluid, and for placement in a mass spectrometer, 10 thereby to determine a test amount of a polypeptide in the sample, wherein the polypeptide is differentially contained in the body fluid of stroke-affected subjects and non-stroke-affected subjects, and has a molecular weight in the range of from 3000 to 30000. BRIEF DESCRIPTION OF THE DRAWINGS 15 Figure 1 (A and B) is a spectral view of plasma from four hemorrhagic stroke patients (H 1-4) and four control samples (CTRL 1-4) using laser desorption/ionization mass spectrometry, in the molecular weight range of 3750 to 4750 Da; 20 Figure 2 (A and B) is a view corresponding to Figure 1, but in the molecular weight range of 5000 to 11000 Da; Figure 3 (A and B) is a view corresponding to Figure 1, but in the molecular weight range of 12000 to 30000 Da; 25 Figure 4 (A and B) is a spectral view of plasma from four ischaemic stroke patients (I 1-4) and four control samples (CTRL 1-4) using laser desorption/ionization mass spectrometry, in the molecular weight range of 3750 to 4750 Da; 30 Figure 5 (A and B) is a view corresponding to Figure 4, but in the molecular weight range of 5000 to 11000 Da; WO 03/069346 PCT/EPO3/01462 -4 Figure 6 (A and B) is a view corresponding to Figure 4, but in the molecular weight range of 12000 to 30000 Da; Figure 7 (A, B and C) is a spectral view of plasma from four stroke patients (identified 5 as 155 stroke, 184 stroke, 194 stroke and 195 stroke) and four control samples (identified as 380 neg, 386 neg, 387 neg and 390 neg) using laser desorption/ionization mass spectrometry, in the molecular weight range of about 4300 to 5000 Da; 10 Figure 8 (A, B and C) is a view corresponding to Figure 7, but in the molecular weight range of about 5000 to 8000 Da; and Figure 9 (A, B and C) is a view corresponding to Figure 7, but in the molecular weight range of 10000 to 16000 Da. 15 In the Figures, the horizontal axis represents molecular weight in Da (m/z ratio), and the vertical axis represents signal intensity, i.e. amount of material having the given molecular weight. 20 DESCRIPTION OF PREFERRED EMBODIMENTS The invention provides a method of diagnosis of stroke or the possibility thereof in a subject suspected of suffering from stroke. A sample of body fluid taken from the subject is subjected to mass spectrometry, to determine the presence or absence in the 25 sample of a polypeptide marker which is differentially contained in the body fluid of stroke-affected subjects and non-affected subjects. The polypeptide marker has a molecular weight in the range of from 3000 to 30000, preferably from 3900 to 29000, and the presence or absence of the marker is indicative of stroke. A particular feature of the invention is that the presence or absence of certain markers can be used to 30 determine whether a diagnosed stroke is of the ischaemic or haemorrhagic type.

WO 03/069346 PCT/EPO3/01462 -5 The term polypeptide includes proteins and protein fragments, as well as peptides modified by the addition of non-peptide residues, e.g. carbohydrates, phosphates, sulfates or any other post-translational modification. 5 The sample may be adsorbed on a probe under conditions which allow binding between the polypeptide and adsorbent material on the probe. The adsorbent material preferably comprises a metal chelating group complexed with a metal ion, and a preferred metal is copper. Prior to detecting the polypeptide, unbound or weakly bound materials on the probe may be removed with a washing solution, thereby 10 enriching the polypeptide in the sample. The sample is preferably adsorbed on a probe having an immobilised metal affinity capture (IMAC) or a strong anion exchange (SAX) surface capable of binding the polypeptide. The sample may be also adsorbed on a probe having hydrophobic, strong anionic or weak cationic exchange surfaces under conditions which allow binding of the polypeptides. The probe may 15 consist of a strip having several adsorbent wells, and be inserted into the spectrometer, then movable therein so that each well is in turn struck by the ionizing means (e.g. laser) to give a spectrometer reading. The polypeptide is preferably determined by surface-enhanced laser desorption/ionisation (SELDI) and time of flight mass spectrometry (TOF-MS). 20 In principle, any body fluid can be used to provide a sample for diagnosis, but preferably the body fluid is cerebrospinal fluid (CSF), plasma, serum, blood, urine or tears. 25 In one embodiment of the invention, one or more polypeptides having a respective molecular weight of about 3900, about 3970, about 3990, about 6945, about 10070, about 14040 and/or about 28000 is determined, and increase or reduction, relative to a control, of peaks corresponding to such polypeptides is indicative of stroke. The 3900 peak is mostly higher than the 3970 and 3990 peaks in stroke plasma samples. 30 In another embodiment of the invention, one or more polypeptides having a respective molecular weight of about 5920, about 6660 and/or about 7770 is determined, and WO 03/069346 PCT/EPO3/01462 -6 increase or reduction, relative to a control, of peaks corresponding to such polypeptides is indicative of stroke. In a further embodiment of the invention, one or more polypeptides having a 5 respective molecular weight of about 3900, about 3970, about 3990, about 14040 and/or about 28000 is determined, and increase or reduction, relative to a control, of peaks corresponding to such polypeptides is used to indicate whether a diagnosed stroke is of the ischaemic or haemorrhagic type. 10 Generally, the following observations, separately or in any combination, are characteristic of haemorrhagic stroke (when compared to a control): decrease of a peak at about 3970; decrease of a peak at about 5920 and/or about 10070; increase of a peak at about 6660 and/or about 6945 and/or about 7770; and decrease of a peak at about 14040 and/or about 28000. 15 Generally, the following observations, separately or in any combination, are characteristic of ischaemic stroke (when compared to a control): a peak at about 3970 greater than a peak at about 3990, but both lower than a peak at about 3900; decrease of a peak at about 5920 and/or about 10070; increase of a peak at about 7770; and no 20 decrease of peaks at about 14040 and/or about 28000. In a further embodiment of the invention, one or more polypeptides having a respective molecular weight of about 4475, about 4634 and/or about 4797 is determined, and reduction, relative to a control, of peaks corresponding to such 25 polypeptides is indicative of stroke. In a still further embodiment, one or more polypeptides having a respective molecular weight of about 6441 and/or about 6643 is determined, and increase, relative to a control, of peaks corresponding to such polypeptides is indicative of stroke. 30 In a yet further embodiment, one or more polypeptides having a respective molecular weight of about 11530 and/or about 11712 is determined, and reduction, relative to a control, of peaks corresponding to such polypeptides is indicative of stroke.

WO 03/069346 PCT/EPO3/01462 -7 According to the invention, a diagnosis of stroke may be made from measurement at a single molecular weight or at any combination of two or more molecular weights, which may, for example, be selected from those molecular weights mentioned above. 5 Measurement of the molecular weight of the polypeptide or polypeptides is effected in the mass spectrometer. The molecular weights quoted above can be measured with an accuracy of better than 1%, and preferably to within about 0.1%. The term "about" in connection with molecular weights therefore means within a variation of about 1%, 10 preferably within about 0.1%, above or below the quoted value. The invention also relates to the use of a polypeptide which is differentially contained in a body fluid of stroke-affected subjects and non-stroke-affected subjects, the polypeptide having a molecular weight in the range of from 3000 to 30000 and being 15 determinable by mass spectrometry, for diagnostic, prognostic and therapeutic applications. This may involve the preparation and/or use of a material which recognizes, binds to or has some affinity to the above-mentioned polypeptide. Examples of such materials are antibodies and antibody chips. The term "antibody" as used herein includes polyclonal antiserum, monoclonal antibodies, fragments of 20 antibodies such as Fab, and genetically engineered antibodies. The antibodies may be chimeric or of a single species. The above reference to "prognostic" applications includes making a determination of the likely course of a stroke by, for example, measuring the amount of the above-mentioned polypeptide in a sample of body fluid. The above reference to "therapeutic" applications includes, for example, preparing 25 materials which recognize, bind to or have affinity to the above-mentioned polypeptides, and using such materials in therapy. The materials may in this case be modified, for example by combining an antibody with a drug, thereby to target the drug to a specific region of the animal to be treated. 30 The methodology of this invention can be applied to the diagnosis of any kind of stroke. Body fluid samples are prepared from stroke-affected and non-stroke-affected subjects. The samples are applied to a probe having a surface treated with a variety of adsorbent media, for differential retention of peptides in the sample, optionally using WO 03/069346 PCT/EPO3/01462 -8 washing liquids to remove unbound or weakly bound materials. If appropriate, energy-absorbing material can also be applied. The probe is then inserted into a mass spectrometer, and readings are taken for the various sample/adsorbent combinations using a variety of spectrometer settings. Comparison of the affected and non-affected 5 samples under a given set of conditions reveals one or more polypeptides which are differentially expressed in the affected and non-affected samples. The presence or absence of these polypeptides can then be used in the testing of a fluid sample from a subject under the same conditions (adsorbent, spectrometer settings etc.) to determine whether or not the subject is affected. Furthermore, by comparing, on the one hand, 10 haemorrhagic stroke samples with a control, and, on the other hand, ischaemic stroke samples with a control, it is possible to discriminate between the possibility of haemorrhagic stroke or ischaemic stroke by testing a body fluid sample from a patient under the same conditions. 15 The above reference to "presence or absence" of a polypeptide should be understood to mean simply that there is a significant difference in the amount of a polypeptide which is detected in the affected and non-affected sample. Thus, the "absence" of a polypeptide in a test sample may include the possibility that the polypeptide is actually present, but in a significantly lower amount than in a comparative test sample. 20 According to the invention, a diagnosis can be made on the basis of the presence or absence of a polypeptide, and this includes the presence of a polypeptide in a significantly lower or significantly higher amount with reference to a comparative test sample. 25 The following Examples illustrate the invention. EXAMPLE 1 The objective of the present study was to detect specific polypeptides in body fluids 30 (cerebrospinal fluid, plasma and others) of stroke-affected patients. Samples were analysed by the Surface Enhanced Laser Desorption Ionization (SELDI) Mass Spectroscopy (MS) teelmology. This technology encompasses micro-scale affinity capture of proteins by using different types of retentate chromatography and then WO 03/069346 PCT/EPO3/01462 -9 analysis by time of flight mass spectrometry. Difference maps are thus generated each corresponding to a typical protein profiling of given samples that were analysed with a Ciphergen Biosystem PBS II mass spectrometer (Freemont, CA, USA). Differential expressed peaks were identified when comparing spectra generated in a group of 5 plasma samples from stroke-affected patients with a control group of non-affected patients. The SELDI analysis was performed using 2pl of crude human plasma samples in order to detect specific polypeptides with metal affinity. An immobilized copper 10 affinity array (IMAC-Cu

++

) was employed in this approach to capture proteins with affinity for copper to select for a specific subset of proteins from the samples. Captured proteins were directly detected using the PBSII Protein Chip Array reader (Ciphergen Biosystems, Freemont, CA, USA). 15 The following protocol was used for the processing and analysis of ProteinChip arrays using Chromatographic TED-Cu(II) adsorbent array. TED is a (tris(carboxymethyl)ethylenediamine-Cu) adsorbent coated on a silicon oxide-coated stainless steel substrate. 20 * The surface was first loaded with 10 ptl of 100 mM copper sulfate to each spot and incubated for 15 minutes in a wet chamber. * The chip was thereafter washed by two quick rinses with deionized water for about 10 seconds to remove the excess unbound copper. * Before loading the samples, the I-MAC 3 array was equilibrated once with 5 ttl 25 of PBS NaCl 0.5 M for 5 minutes. * After removing the equilibration buffer, 3 pl of the same buffer were added before applying 2 gl of plasma. The chip was incubated for 20 minutes in a wet chamber. * The samples were thereafter removed and the surface was washed three times 30 with the equilibration buffer (5 minutes each). * Two quick final rinses with water were performed.

WO 03/069346 PCT/EPO3/01462 - 10 * The surface was allowed to air dry, followed by the addition of 0.5 pl of saturated sinapinic acid (SPA, Ciphergen Biosystem) prepared in 50% acetonitrile, 0.5% trifluoroacetic acid. * The chip was air dried again before analysis of the retained protein on each 5 spot with laser desorption/ionization time-of-flight mass spectrometry. * The protein chip array was inserted into the instrument and analysed once the appropriate detector sensitivity and laser energy have been established to automate the data collection. * The obtained spectra were analysed with the Biomark Wizard software 10 (Ciphergen Biosystems, Freemont, CA, USA) running on a Dell Dimension 4100 PC. It generates consistent peak sets across multiple spectra. The results of the above tests on four plasma samples from haemorrhagic stroke patients (plasma H 1-4) and four plasma samples from non-affected subjects (plasma 15 CTRL 1-4) are shown in Figures 1 to 3. Figure 1 shows the strong decrease of a peak around 3970 Da in haemorrhagic samples as compared to healthy ones. In the control samples it forms a pair with a peak at about 3990, but in the haemorrhagic stroke samples the pair have nearly disappeared behind the peak at about 3900, which has been strongly increased. Figure 2 highlights the decrease of two peaks around 5920 20 and 10070 in haemorrhagic stroke samples as compared to healthy ones. Figure 2 also shows the increase of peaks at about 6660, 6945 and 7770 Da in haemorrhagic stroke samples as compared to healthy ones. Figure 3 shows a decreased intensity of peaks at about 14040 and 28000 Da in haemorrhagic stroke samples as compared to healthy ones. 25 EXAMPLE 2 The procedure of Example 1 is repeated on four plasma samples from ischaemic stroke patients (plasma I 1-4) and four plasma samples from non-affected subjects 30 (plasma CTRL 1-4). The results are shown in Figures 4 to 6. Figure 4 shows for the ischaemic stroke samples a pair of peaks at 3970 and 3990, where the 3970 peak is higher than the 3990 peak, but of a lower intensity than the 3900 peak, in contrast to the control samples. Figure 5 highlights the decrease of two peaks around 5920 and WO 03/069346 PCT/EPO3/01462 - 11 10070 in ischaemic stroke samples as compared to healthy ones. Figure 5 also shows the 7770 peak increased in ischaemic stroke samples, but to a lesser extent than in haemorrhagic stroke samples. Figure 6 does not show any decrease of peaks around 14040 and 28000 Da between ischaemic stroke samples and healthy samples, in 5 contrast to the differences shown for haemorrhagic stroke samples in Figure 3. EXAMPLE 3 A comparative investigation between plasma samples coming from 21 stroke patients 10 (including 10 haemorrhagic, 10 ischaemic and 1 unknown type) and 21 healthy patients was carried out using the SELDI technology, in a similar way to the procedure of Example except for the variations mentioned hereafter. SAX ProteinChips (Ciphergen) and a SPA (Ciphergen) matrix were retained for the study. An example of 4 stroke spectra and 4 healthy patient spectra among the 42 tested is 15 given in Figures 7 to 9. Using the Biomarker Wizard (Mann and Whitney statistical analysis), seven peaks appeared differentially expressed between stroke and healthy controls: a decrease of the signal of the peaks at 4475 Da, 4634 Da and 4797 Da is indicative of stroke with p values of 0.000138, 0.00224 and 0.0132 respectively. An increase of the peaks at 6443 Da and 6641 Da is indicative of stroke with p values of 20 0.08950 and 0.02134. And a decrease of the peaks at 11530 Da and 11712 Da, relative to a control, is indicative of stroke with p values of 0.00634 and 0.04034 respectively. The following protocol was used for the processing and analysis of the SAX 25 ProteinChips : 1. Outine each spot using a hydrophobic pen. Allow to dry air 2. Apply 10 pl binding buffer (20 mM Tris - 5 mM NaCl pH9.0) to each spot and incubate in a humidity chamber at room temperature for 5 minutes. Do not allow the spots to become dry. 30 3. Remove excess buffer from the spots without touching the active surface. Repeat steps 2 and 3 two more times.

WO 03/069346 PCT/EPO3/01462 - 12 4. Load 1 gl crude plasma sample + 2 pl binding buffer (20 mM Tris - 5 mM NaCI pH9.0) 5. Incubate in a humidity chamber for 30 minutes. 6. Wash each spot with 5 pl binding buffer (20 mM Tris - 5 mM NaCl pH9.0) 5 5 times, followed by two quick washes with water (5 pl per wash). 7. Wipe dry around the spots. Apply 0.5 p1l SPA saturated matrix (Ciphergen) to each spot while it is still moist, but not wet. Air dry. Apply a second 0.5 p1 of SPA saturated matrix (Ciphergen) and air dry again before analysis of the retained protein on each spot with laser desorption/ionization time-of-flight 10 mass spectrometry 8. The protein chip array was inserted into the instrument and analysed once the appropriate detector sensitivity and laser energy have been established to automate the data collection. 15 Each of the above cited publications is herein incorporated by reference to the extent to which it is relied on herein. 20

Claims (21)

1. A method of diagnosis of stroke or the possibility thereof in a subject suspected of suffering from stroke, which comprises subjecting a sample of body fluid 5 taken from the subject to mass spectrometry, thereby to determine a test amount of a polypeptide in the sample, wherein the polypeptide is differentially contained in the body fluid of stroke-affected subjects and non-stroke-affected subjects, and has a molecular weight in the range of from 3000 to 30000; and determining whether the test amount is consistent with a diagnosis of stroke. 10

2. A method according to Claim 1, in which the polypeptide is present in the body fluid of stroke-affected subjects and not present in the body fluid of non-stroke affected subjects, whereby the presence of the polypeptide in a body fluid sample is indicative of stroke. 15

3. A method according to Claim 1, in which the polypeptide is not present in the body fluid of stroke-affected subjects and present in the body fluid of non-stroke affected subjects, whereby the non-presence of the polypeptide in a body fluid sample is indicative of stroke. 20

4. A method according to any of Claims 1 to 3, in which the mass spectrometry is laser desorption/ionization mass spectrometry.

5. A method according to any of Claims 1 to 4, in which the sample is adsorbed 25 on a probe having an immobilised metal affinity capture (IMAC), hydrophobic, strong anionic or weak cationic exchange surface capable of binding the polypeptide.

6. A method according to any of Claims 1 to 5, in which the polypeptide is determined by surface-enhanced laser desorption/ionisation (SELDI) and time of 30 flight mass spectrometry (TOF-MS).

7. A method according to any of Claims 1 to 6, in which the body fluid is cerebrospinal fluid, plasma, serum, blood or tears. WO 03/069346 PCT/EPO3/01462 - 14

8. A method according to any of Claims 1 to 7, in which a plurality of peptides is determined in the sample.

9. A method according to any of Claims 1 to 8, in which the test amount of 5 polypeptide is used to determine whether a diagnosed stroke is of the ischaemic or haemorrhagic type.

10. A method according to any of Claims 1 to 9, in which one or more polypeptides having a respective molecular weight of about 3900, about 3970, about 10 3990, about 6945, about 10070, about 14040 and/or about 28000 is determined, and increase or reduction, relative to a control, of peaks corresponding to such polypeptides is indicative of stroke.

11. A method according to any of Claims 1 to 10, in which one or more 15 polypeptides having a respective molecular weight of about 5920, about 6660 and/or about 7770 is determined, and increase or reduction, relative to a control, of peaks corresponding to such polypeptides is indicative of stroke.

12. A method according to any of Claims 1 to 11, in which one or more 20 polypeptides having a respective molecular weight of about 3900, about 3970, about 3990, about 14040 and/or about 28000 is determined, and increase or reduction, relative to a control, of peaks corresponding to such polypeptides is used to indicate whether a diagnosed stroke is of the ischaemic or haemorrhagic type. 25

13. A method according to any of Claims 1 to 12, in which one or more polypeptides having a respective molecular weight of about 4475, about 4634 and/or about 4797 is determined, and reduction, relative to a control, of peaks corresponding to such polypeptides is indicative of stroke. 30

14. A method according to any of Claims 1 to 13, in which one or more polypeptides having a respective molecular weight of about 6441 and/or about 6643 is determined, and increase, relative to a control, of peaks corresponding to such polypeptides is indicative of stroke. WO 03/069346 PCT/EPO3/01462 - 15

15. A method according to any of Claims 1 to 14 in which one or more polypeptides having a respective molecular weight of about 11530 and/or about 11712 is determined, and reduction, relative to a control, of peaks corresponding to such 5 polypeptides is indicative of stroke.

16. Use of a polypeptide which is differentially contained in a body fluid of stroke affected subjects and non-stroke-affected subjects, the polypeptide having a molecular weight in the range of from 3000 to 30000 and being determinable by mass 10 spectrometry, for diagnostic, prognostic and therapeutic applications.

17. Use for diagnostic, prognostic and therapeutic applications of a material which recognizes, binds to or has affinity for a polypeptide which is differentially contained in a body fluid of stroke-affected subjects and non-stroke-affected subjects, the 15 polypeptide having a molecular weight in the range of from 3000 to 30000 and being determinable by mass spectrometry.

18. Use according to Claim 17, in which the material is an antibody or antibody chip. 20

19. A kit for use in diagnosis of stroke, comprising a probe for receiving a sample of body fluid, and for placement in a mass spectrometer, thereby to determine a test amount of a polypeptide in the sample, wherein the polypeptide is differentially contained in the body fluid of stroke-affected subjects and non-stroke-affected 25 subjects, and has a molecular weight in the range of from 3000 to 30000.

20 A kit according to Claim 19, in which the probe contains an adsorbent for adsorption of the polypeptide. 30

21. A kit according to Claim 20, further comprising a washing solution for removal of unbound or weakly bound materials from the probe.