JP2005502882A - Diagnosis of infectious spongiform encephalopathy - Google Patents

Abstract

Objective: To diagnose infectious spongiform encephalopathy (TSE) using mass spectrometry to observe a polypeptide in a sample of body fluid obtained from a subject. Polypeptides are contained differently in the body fluids of TSE infected and non-infected subjects and have a molecular weight in the range of 3500-30000.

Description

【Technical field】
[0001]
The present invention relates to a method for diagnosing infectious spongiform encephalopathy (TSE).
[Background]
[0002]
Infectious spongiform encephalopathy (TSEs) is a neurodegenerative disease of the central nervous system. They can be transmitted, inherited or sporadic, and are also observed in animals, such as bovine spongiform encephalopathy (BSE) in cattle or scrapie in sheep, Creutzfeldt-Jakob in humans Similar to illness (CJD), Gerstmann-Stroisler-Scheinker syndrome, fatal familial insomnia, or cool. They have a long incubation period and lead to ataxia, dementia, mental disorders, and death. Neuropathic changes include vacuolar degeneration of brain tissue, astrogliosis, and amyloid plaque formation. It is difficult to diagnose that these diseases develop before birth.
DISCLOSURE OF THE INVENTION
[Problems to be solved by the invention]
[0003]
CJD patient cerebrospinal fluid (CSF) was analyzed by two-dimensional polyacrylamide gel electrophoresis [Harrington, M .; G. New England Journal of Medicine 315, 279 (1986), Hsick, G. et al. Kenney, K .; , Gibbs, C.I. J. et al. Lee, K .; H. & Harrington, M .; B. New England Journal of Medicine 335, 924 (1996). ] Shows two added polypeptides (known as 14-3-3 polypeptides). The effect of those 14-3-3 polypeptides remains unclear in TSE. They can be used in prenatal testing due to advances in CJD diagnosis, but are less specific.
[0004]
PCT specifications WO 98/23962 and 98/32710; Schmerr, M .; J. et al. , The Beckmann Coulter Pace Setter Newsletter 3 (2), 1-4 (June 1999), the use of monoclonal antibodies against prion protein aberrant formation (related to CJD) Can be used in enzyme immunoassays, but these methods have not yet been fully developed.
[0005]
PCT / EP 01/02894 relates to diagnostic analysis for TSEs in which the concentration of cardiac or brain fatty acids bound to a protein (H-FABP or B-FABP) is determined in a sample of body fluid.
[0006]
US-A-6225047 describes the use of retentate chromatography to generate a difference map, and in particular describes a method for identifying analytes that differ between two samples. One special method described therein is laser desorption mass spectrometry.
[0007]
WO 01/25791 describes a method for assisting in the diagnosis of prostate cancer, which determines the test amount of a polypeptide marker, which is a subject of prostate cancer patients and non-prostate cancer patients. There are differences in the samples. The marker is determined using mass spectrometry, preferably laser desorption mass spectrometry.
[0008]
New methods for determining the development and markers of new non-invasive TSE markers for body fluids (especially CJD and BSE markers in blood) will help clinicians establish early diagnosis. This problem is now solved by the present invention.
[Means for Solving the Problems]
[0009]
The present invention provides a method for diagnosing infectious spongiform encephalopathy (TSE), or a method for diagnosing that possibility in a subject suspected of having TSE. The diagnostic method consists of mass analyzing a sample of bodily fluid removed from the subject, thereby determining the test amount of the polypeptide in the sample. The polypeptide is contained differently in the body fluids of TSE infected and non-TSE infected subjects, has a molecular weight in the range of 3500 to 30000, and is the test amount consistent with the diagnosis of TSE? Determined by no.
[0010]
The present invention is also a polypeptide that is differentially contained in the body fluids of TSE-infected subjects and non-TSE-infected subjects, and has a molecular weight in the range of 3500 to 30000, and is diagnostic, predictive, therapeutic For applications, the use of polypeptides that can be determined by mass spectrometry is provided.
[0011]
The present invention further provides kits for use in the diagnosis of TSE. The kit consists of a probe for receiving a sample of body fluid and placing it by mass spectrometry, thereby determining the test amount of the polypeptide in the sample. The polypeptide is contained differently in body fluids between TSE-infected subjects and non-TSE-infected subjects and has a molecular weight in the range of 3500 to 30000.
BEST MODE FOR CARRYING OUT THE INVENTION
[0012]
The present invention provides a method for diagnosing infectious spongiform encephalopathy (TSE), or a method for diagnosing that possibility in a subject suspected of having TSE. A sample of body fluid removed from the subject is subjected to mass spectrometry to determine the presence or absence of a polypeptide marker contained differently in the body fluid of the TSE infected subject and the uninfected subject. The polypeptide marker has a molecular weight in the range of 3500 to 30000, preferably 3900 to 18000, which guides whether the presence or absence of the marker is TSE.
[0013]
This method can be applied to all types of TSE and can be applied to any human or animal suffering from or suspected of suffering. This method is particularly applicable to the diagnosis of symptoms similar to BSE in other animals, such as CJD in human patients, especially new mutant CJD, BSE in ruminants such as cattle, and scrapie in sheep.
[0014]
The term polypeptide includes proteins and protein fragments as well as peptides modified by, for example, the addition of non-peptide residues of carbohydrates, phosphates, sulfates, or other post-translational modifications.
[0015]
The sample is absorbed by the probe under conditions that allow binding between the polypeptide and the absorbent material of the probe. The absorbent material is preferably composed of metal chelate groups that complex with metal ions, and the preferred metal is copper. Prior to detection of the polypeptide, material that is unbound or weakly bound to the probe is removed with a wash solution, thereby enriching the polypeptide in the sample. The sample is preferably absorbed by a probe having an immobilized metal affinity capture (IMAC) surface that can bind to the polypeptide. The sample is also absorbed by a probe having a hydrophobic, strong anionic, or weakly cationic exchange surface under conditions that allow the polypeptide to bind. The probe consists of an elongated piece with several suction holes, placed in the spectrometer, then moved in it, each piece is struck in turn by the ionization means (eg laser) and read out by the spectrometer . The polypeptide is preferably determined by surface amplified laser desorption / ionization (SELDI) and time-of-flight mass spectrometry (TOF-MS).
[0016]
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. is there.
[0017]
In one embodiment of the invention, the TSE is Creutzfeldt-Jakob disease (CJD). In this case, the polypeptide preferably has a molecular weight of about 4780, about 6700, about 8600, or about 13375, indicating that one or more of such polypeptides is CJD. Alternatively, one or more polypeptides each having a molecular weight of about 3970, about 3990, about 4294, about 4478, about 10075, about 11730, about 14043, or about 17839 are determined, and so on One or more deficiencies of a particular polypeptide is shown to be CJD. Further alternatively, a polypeptide having a molecular weight of about 7700 is determined, indicating that the presence of such a polypeptide is CJD. For another example, CJD has one or more of the following peaks: about 3295, about 4315, about 4436, about 6200, about 8936, about 9107, about 9145, about 9185, about 9454, and about Indicated by a decrease of 13550 Da. For further examples, CJD is also indicated by an increase in one or more of the following peaks: about 7574, about 7930, about 7975, and 8020. The present invention finds its true value in adopting measurement at any one or more of the above molecular weight values in any combination thereof for the purpose of diagnosing CDJ.
[0018]
In another embodiment of the invention, the TSE is bovine spongiform encephalopathy (BSE). In this case, the polypeptide preferably has a molecular weight of about 10220, indicating that the presence of the polypeptide is BSE.
[0019]
In a further embodiment of the invention, the TSE is scrapie.
Measurement of the molecular weight of a polypeptide or group of polypeptides is accomplished by mass spectrometry. All molecular weights here are measured by Da. The molecular weight quoted above can be measured with an accuracy better than 1%, preferably within the range of about 0.1%. As used herein, the term “about” in terms of molecular weight means within a range of about 1% variation, preferably within a range of about 0.1%, for the values quoted above or below.
[0020]
The present invention also relates to the use of a polypeptide that is differentially contained in the body fluids of TSE infected and non-infected subjects for diagnostic, predictive and therapeutic applications, the polypeptide having a range of 3500 to 30000. It has a molecular weight and can be determined by mass spectrometry. This involves the synthesis and / or use of materials that recognize, bind to or have some affinity for the polypeptides referred to above. Examples of such materials are antibodies and antibody chips. The term “antibody” as used herein includes polyclonal antisera, monoclonal antibodies, fragments of antibodies such as Fab, and genetically modified antibodies. The antibody may be a chimera or a single species. The “predictive” application referred to above includes determining the likely course of TSE, for example by measuring the amount of the polypeptide referred to above in a sample of body fluid. The “therapeutic” applications referred to above include, for example, the synthesis of materials that recognize, bind to, or have an affinity for the polypeptides referred to above, and do so in therapy. The use of new materials. The material is then modified, for example, by combining an antibody and a drug, thereby targeting the drug to a specific region of the target animal.
[0021]
The method procedure of the present invention can be applied to any TSE diagnosis. Body fluid samples are procured from infected and non-infected subjects. Due to the differential retention of the polypeptide in the sample, the sample was treated with various absorbent media, selectively using wash solution to remove unbound or weakly bound material. It can be applied to a probe having a surface. If appropriate, energy absorbing materials can also be applied. The probe is then entered into mass spectrometry and readings are taken for various sample / absorption combinations using various mass spectrometry settings. Comparison of infected and non-infected samples under a given set of conditions reveals one or more polypeptides that appear differently in infected and non-infected samples. The presence or absence of these polypeptides is used in testing a subject's fluid sample under similar conditions (absorbent, analyzer settings, etc.) that determine whether the subject is infected.
[0022]
It should be understood that reference herein to “present or not” of a polypeptide simply means that there is a considerable difference in the amount of polypeptide detected in a sample of infected and non-infected. is there. Thus, the “presence” of a polypeptide in a test sample includes the possibility that the polypeptide is actually present, but in a significantly lower amount than in the comparative test sample. According to the present invention, a diagnosis is made based on the presence or absence of a polypeptide, which includes the presence of the polypeptide in a significantly smaller or significantly larger amount with reference to a comparative test sample.
[0023]
The following example demonstrates the invention.
[Example 1]
[0024]
The purpose of this study is to detect specific polypeptides in body fluids (cerebrospinal fluid, plasma, and others) of patients with Creutzfeldt-Jakob disease. Samples are analyzed by surface amplified laser desorption ionization (SELDI) mass spectrometry (MS) techniques. This technique involves microscale affinity capture of proteins by using different forms of retentate chromatography and then analysis by time-of-flight mass spectrometry. A different map is generated corresponding to each of the typical protein profilings of a given sample analyzed using Ciphergen Biosystems PBS II mass spectrometry (Fremont, CA, USA). When the spectrum generated in a group of cerebrospinal fluid (CSF) samples from a CJD patient is compared with the sample spectrum generated in a group of demented patients, the peaks that appeared differently are identified.
[0025]
SELDI analysis is performed using 2 μl of an unpurified human CSF sample to detect specific polypeptides with metal affinity. An immobilized copper affinity array (IMAC-Cu ⁺⁺ ) is used in this approach to capture proteins that have an affinity for copper to select for a specific subset of proteins from a sample. Captured proteins are detected directly using a PBSII protein chip array reader (Ciphergen Biosystems, Fremont, CA, USA).
[0026]
The following procedure is used for processing and analysis of protein chip arrays using chromatographic TED-Cu (II) adsorption arrays. TED is an adsorbent (tris (carbonylmethyl) ethylenediamine-Cu) coated on a stainless steel substrate coated with silicon oxide.
The surface is first loaded with 10 μl of 100 mM copper sulfate for each spot and incubated for 15 minutes in a moisturizer.
-The tip is then rinsed by rinsing twice with deionized water for about 10 seconds to remove excess unbound copper.
Before loading the sample, the I-MAC3 array is equilibrated once with 5 μl of PBS NaCl 0.5M for 5 minutes.
After removing the equilibration buffer, add 3 μl of the same buffer before applying 2 μl of CSF. The chip is kept warm in a moisturizing tank for 20 minutes.
The sample is then removed and the surface is washed 3 times with equilibration buffer (every 5 minutes).
・ Pour with water twice at the end.
The surface is air-dried and then 0.5 μl of unsaturated sinapinic acid (SPA, Ciphergen Biosystem) synthesized in 50% acetonitrile and 0.5% trifluoroacetyl acid is added.
• Air dry the chip again before analysis of retained protein in each spot with laser desorption / ionization time-of-flight mass spectrometry.
• Once the appropriate detection sensitivity and laser energy are established to automate data collection, the protein chip array is placed into the instrument and analyzed.
The resulting spectrum is analyzed with Biomark Wizard software (Ciphergen Biosystems, Fremont, CA, USA) running on a Dell Dimension 4100 PC. It produces a peak set that crosses and matches multiple spectra.
[0027]
FIGS. 1-4 show the results of a comparative study conducted between CSF from CJD diagnosed patients and standard CSF using the IMAC3 protein chip array prepared as described above. In this study, we found that the four peaks had a significantly different increase in CSF from patients affected by CJD. Their molecular weights are about 4780, 6700, 8600, and 13375, respectively (mass accuracy is about 0.1%). FIG. 1 shows two spectral views from 0 to 100,000 Da, showing a standard sample and a CJD sample, respectively. FIG. 2 shows the protein peak at 4780 Da. FIG. 3 shows the protein peaks at 6700 and 8600 Da. FIG. 4 shows the protein peak at 13375 Da. These data demonstrate that the peaks at about 4780, 6700, 8600, and 13375 Da can be used to diagnose CJD in CSF samples.
[Example 2]
[0028]
Example 1 is repeated using plasma samples from BSE infected cattle (BSE +) and non-infected cattle (BSE-). The result is shown in FIGS. FIG. 5 shows a spectral view for each type of sample from 0 to 50,000 Da. We observed that the protein of about 10220 Da was significantly increased in the BSE + plasma sample as illustrated in FIG. This demonstrates that the peak at about 10220 Da can be used to diagnose BSE in plasma samples.
[Example 3]
[0029]
Example 2 is repeated using plasma samples from CJD infected patients (CJD +) and non-infected patients (CJD-, also referred to as CTS = Swiss Transfusion Center). This result is shown in FIGS. FIG. 7 shows a spectral view for each type of sample from 0 to 50,000 Da. We have found that about 3970, about 3990, about 4294, about 4478, about 10075, about 11730, about 14043, or about 17839 polypeptides are significantly reduced in CJD + plasma samples as described in FIGS. 8A and 8B. Observed that. We also observed that the peak at about 7770 Da increased in the CJD + plasma sample, as illustrated in FIG. 8B. This means that peaks of about 3970, about 3990, about 4294, about 4478, about 10075, about 11730, about 14043, about 17839, or about 7770 Da can be used to diagnose CJD in plasma samples. Prove that.
[Example 4]
[0030]
Example 3 is repeated, but using a more recent version of the software that analyzes the data. The results are shown in FIGS. 9A through 9E, showing some new variations at the protein level in addition to their identification in the above examples.
[0031]
In FIG. 9A, the arrow indicates a peak at about 3295 Da, which peak decreases in the CJD sample.
In FIG. 9B, numbered arrows indicate the following.
[0032]
1—a peak at about 3976 Da, which is reduced in the CJD sample (corresponding to the 3970 Da peak in Example 3).
2—A peak at about 3992 Da, which is reduced in the CJD sample (corresponding to the 3990 Da peak in Example 3).
[0033]
3—A peak at about 4300 Da, which is reduced in the CJD sample (corresponding to the 4294 Da peak in Example 3).
4—A peak at about 4315 Da, which decreases in the CJD sample.
[0034]
5—A peak at about 4436 Da, which is reduced in the CJD sample.
6—A peak at about 4484 Da, which is reduced in the CJD sample (corresponding to the 4478 Da peak in Example 3).
[0035]
In FIG. 9C, the arrow indicates a peak at about 6200 Da, which peak decreases in the CJD sample.
In FIG. 9D, numbered arrows indicate the following.
[0036]
10—about 7574 Da peak, increasing in the CJD sample.
11—A peak at about 7773 Da, which is increased in the CJD sample (corresponding to the 7770 Da peak in Example 3).
[0037]
12—a peak at about 7930 Da, which is increased in the CJD sample.
13—A peak at about 7975 Da, which is increased in the CJD sample.
[0038]
14—A peak at about 8020 Da, which increases in the CJD sample.
15—about 8936 Da peak, which is reduced in the CJD sample.
[0039]
16—A peak at about 9107 Da, which is reduced in the CJD sample.
17—A peak at about 9145 Da, which is reduced in the CJD sample.
[0040]
18—A peak at about 9185 Da, which is reduced in the CJD sample.
19—A peak at about 9454 Da, which is reduced in the CJD sample.
[0041]
In FIG. 9E, numbered arrows indicate the following.
20—about 10068 Da peak, which is reduced in the CJD sample (corresponding to the 10075 Da peak in Example 3).
[0042]
21—about 13550 Da peak, which is reduced in the CJD sample.
22—about 17809 Da peak, which is reduced in the CJD sample (corresponding to the 17839 Da peak in Example 3).
[0043]
This example demonstrates that any one of the above peaks, or any combination of two or more of them can be used to diagnose CJD.
Each of the publications cited above is incorporated herein by reference to the extent dependent on this specification.
[Brief description of the drawings]
[0044]
FIG. 1 is a spectral diagram of CSF from a standard sample and a sample of CJD infection using laser desorption / ionization mass spectrometry.
FIG. 2 is a correspondence diagram highlighting the protein peak at about 4780 Da in CJD infected CSF samples.
FIG. 3 is a correspondence diagram highlighting protein peaks at approximately 6700 and 8600 Da in CJD-infected CSF samples.
FIG. 4 is a correspondence diagram highlighting the protein peak at about 13375 Da in CJD infected CSF samples.
FIG. 5 is a spectral diagram of plasma from a standard sample and a BSE infected sample using laser desorption / ionization mass spectrometry.
FIG. 6 corresponds to FIG. 5 and highlights the protein peak at approximately 10220 Da in the BSE-infected plasma sample.
FIG. 7 is a spectral diagram of plasma from CJD infected patients (CJD +) and non-CJD infected patients (CJD−) using laser desorption / ionization mass spectrometry.
FIG. 8A is a diagram corresponding to FIG. 7 and highlighting polypeptide peaks that are expressed differently in CJD + and CJD− plasma samples.
FIG. 8B is a diagram corresponding to FIG. 7 and highlighting polypeptide peaks that are differentially represented in plasma samples of CJD + and CJD−.
FIG. 9A shows plasma from CJD-infected patients (plasma CJD) and plasma from non-infected patients (plasma CTS) further highlighting the differentially expressed polypeptide peaks in infected and non-infected samples. FIG.
FIG. 9B shows plasma from CJD-infected patients (plasma CJD) and plasma from non-infected patients (plasma CTS) further highlighting the differentially expressed polypeptide peaks in infected and non-infected samples. FIG.
FIG. 9C shows plasma from CJD-infected patients (plasma CJD) and plasma from non-infected patients (plasma CTS) further highlighting the differentially expressed polypeptide peaks in infected and uninfected samples. FIG.
FIG. 9D shows plasma from CJD-infected patients (plasma CJD) and plasma from non-infected patients (plasma CTS) further highlighting the differentially expressed polypeptide peaks in infected and uninfected samples. FIG.
FIG. 9E shows plasma from CJD-infected patients (plasma CJD) and plasma from non-infected patients (plasma CTS) further highlighting the differentially expressed polypeptide peaks in infected and uninfected samples. FIG.

Claims (23)

Infectious spongiform encephalopathy (TSE) or a method of diagnosing that possibility in a subject suspected of suffering from the TSE is:
Subjecting a sample of body fluid removed from the subject to mass spectrometry, thereby determining a test amount of the polypeptide in the sample;
Determining whether the test quantity corresponds to a diagnosis of the TSE;
The polypeptide is contained differently in the body fluids of TSE-infected subjects and non-infected subjects and has a molecular weight in the range of 3500 to 30000. 2. The polypeptide of claim 1, wherein the polypeptide is present in the body fluid of a TSE infected subject and not in the body fluid of a non-TSE infected subject, thereby indicating that the presence of the polypeptide in a body fluid sample is TSE. Method. 2. The polypeptide is not present in the body fluid of a TSE-infected subject but is present in the body fluid of a non-TSE-infected subject, thereby indicating that the absence of the polypeptide in the body fluid sample is TSE. the method of. The method according to claim 1, wherein the mass spectrometry is laser desorption / ionization mass spectrometry. 5. The sample of claim 1, wherein the sample is absorbed with a probe having immobilized metal affinity capture (IMAC), a hydrophobic, strong anionic, or weakly cationic exchange surface that binds to the polypeptide. The method according to item. 6. The method of any one of claims 1-5, wherein the polypeptide is determined by surface amplified laser desorption / ionization (SELDI) and time-of-flight mass spectrometry (TOF-MS). The method according to any one of claims 1 to 6, wherein the body fluid is cerebrospinal fluid, plasma, serum, blood, or tears. 8. The method of any one of claims 1 to 7, wherein a plurality of the polypeptides are determined within the sample. The method according to any one of claims 1 to 8, wherein the TSE is Creutzfeldt-Jakob disease (CJD). One or more respective polypeptides having a molecular weight of about 4780, about 6700, about 8600, or about 13375 are determined, indicating that the presence of one or more of such polypeptides is CJD The method of claim 9. One or more polypeptides each having a molecular weight of about 3970, about 3990, about 4294, about 4478, about 10075, about 11730, about 14043, or about 17839 are determined, and one or more of such polypeptides 11. A method according to claim 9 or 10, wherein a greater absence indicates CJD. 12. A method according to any one of claims 9 to 11 wherein a polypeptide having a molecular weight of about 7700 is determined and the presence of such a polypeptide is CJD. A polypeptide having a molecular weight of about 3295, about 4315, about 4436, about 6200, about 8936, about 9107, about 9145, about 9185, about 9454, or about 13550 Da is determined, and one or more of such polypeptides 13. A method according to any one of claims 9 to 12, wherein the absence or reduction amount indicates CJD. The polypeptide of claim 9 to 13, wherein a polypeptide having a molecular weight of about 7574, about 7930, about 7975, or about 8020 Da is determined and indicates that the presence or increase of one or more of such polypeptides is CJD The method according to any one of the above. The method according to any one of claims 1 to 8, wherein the TSE is bovine spongiform encephalopathy (BSE). 16. The method of claim 15, wherein the polypeptide has a molecular weight of about 10220, wherein the presence of the polypeptide is BSE. 9. A method according to any one of claims 1 to 8, wherein the TSE is scrapie. Use of a polypeptide that is differentially contained in the body fluids of a TSE-infected subject and a non-infected subject for diagnostic, predictive, therapeutic applications,
The polypeptide has a molecular weight in the range of 3500 to 30000 and can be determined by mass spectrometry. The use of materials for diagnostic, predictive, therapeutic applications,
The material recognizes a polypeptide contained differently in the body fluid of a TSE-infected subject and a non-infected subject, binds to the polypeptide, or has an affinity for the polypeptide,
The prepolypeptide has a molecular weight in the range of 3500 to 30000 and can be determined by mass spectrometry. The use according to claim 19, wherein the material is an antibody or an antibody chip. A kit for use in diagnosis of TSE comprising a probe for receiving a sample of body fluid and placing it in mass spectrometry;
Thereby determining the test amount of polypeptide in the sample,
The polypeptide is contained differently in body fluids between TSE-infected subjects and non-TSE-infected subjects,
The polypeptide has a molecular weight in the range of 3500 to 30000. The kit of claim 21, wherein the probe comprises an adsorbent for absorption of the polypeptide. 23. The kit of claim 22, further comprising a wash solution for removing unbound or weakly bound material from the probe.