DIAGNOSTIC METHOD FOR MULTIPLE SCLEROSIS

Abstract

A method of detecting the presence, or monitoring the severity of a condition characterised by the presence of fragments of a marker protein in the brain of a patient. The method comprises: (i) providing a sample comprising macrophages obtained from the patient; and (ii) detecting the presence of the marker protein or fragments thereof in the macrophages. The presence of abnormal levels of the marker protein and/or fragments thereof in the macrophages is indicative of the presence of the condition in the patient. The condition and the marker proteins can be: Alzheimer's Disease and the Abeta peptide, Parkinson's Disease and ubiquitin, Multiple Sclerosis and myelin basic protein, FrontoTemporal Dementia and tau, Amyotrophic Lateral Sclerosis and tau, Parkinson's disease, Lewy Body dementia or Alzheimer's Disease and alpha-synuclein.

Claims

1-35. (canceled)

36. A method of detecting the presence of or monitoring the severity of a disease condition in a patient comprising: providing a blood or cerebro-spinal fluid (CSF) sample from the patient; and detecting the presence of (1) a marker protein or fragments of the marker protein in activated macrophages of the blood or CSF sample and (2) the presence of an additional marker specifically in the blood or CSF sample, wherein both abnormal levels of the marker protein or fragments of the marker protein in the activated macrophages and the presence of the additional marker in the blood or CSF sample are indicative of the presence of or severity of the disease condition in the patient.

37. The method of claim 36, wherein the marker protein is Ali peptide.

38. The method of claim 37, wherein the disease condition is Alzheimer's Disease.

39. The method of claim 38, wherein the additional marker comprises abnormal levels of Aβ42, Tau, Phospho-Tau, Abeta42/Abeta40 ratio, or combinations thereof, in the CSF sample of the patient.

40. The method of claim 38, wherein the abnormal levels of the additional marker comprise a concentration of Aβ42 in the CSF sample of the patient of less than 550 pg/mL.

41. The method of claim 38, wherein the abnormal levels of the additional marker comprise a concentration of Phospho-Tau in the CSF sample of the patient of greater than 85 pg/mL.

42. The method of claim 38, wherein the abnormal levels of the additional marker comprise a concentration ten times the Abeta42/Abeta40 ratio in the CSF sample of the patient of less than one.

43. The method of claim 36, further comprising comparing the levels of the marker protein or fragments of the marker protein with a standard level, the standard level being an average of the levels of the marker protein or fragments of the marker protein in macrophages obtained from a plurality of individuals without the condition, wherein the levels of the marker protein or fragments of the marker protein are abnormal if there is a statistically significant difference from the standard level.

44. The method of claim 36, wherein the disease condition is Parkinson's Disease, Multiple Sclerosis, FrontoTemporal Dementia, or Amyotrophic Lateral Sclerosis.

45. The method of claim 36, wherein the marker protein is ubiquitin, myelin basic protein, tau protein, or alpha-synuclein.

46. A method of detecting the presence of or monitoring the severity of a disease condition in a patient comprising: providing a cerebro-spinal fluid (CSF) or a blood sample from the patient; and detecting the presence of (1) a marker protein or fragments of the marker protein in activated macrophages of the blood or CSF sample and (2) the presence of an additional marker specifically in an RNA profile of blood or CSF obtained from the patient, wherein both abnormal levels of the marker protein or fragments of the marker protein in the activated macrophages and the presence of the additional marker in the RNA profile are indicative of the presence of or severity of the disease condition in the patient.

47. The method of claim 46, wherein the marker protein is Aβ peptide.

48. The method of claim 47, wherein the disease condition is Alzheimer's Disease.

49. The method of claim 48, wherein the additional marker comprises abnormal levels of Aβ42, Tau, Phospho-Tau, Abeta42/Abeta40 ratio, or combinations thereof, in the CSF sample of the patient.

50. The method of claim 48, wherein the abnormal levels of the additional marker comprise a concentration of Aβ42 in the RNA profile of the patient of less than 550 pg/mL.

51. The method of claim 48, wherein the abnormal levels of the additional marker comprise a concentration of Phospho-Tau in the RNA profile of the patient of greater than 85 pg/mL.

52. The method of claim 48, wherein the abnormal levels of the additional marker comprise a concentration ten times the Abeta42/Abeta40 ratio in the RNA profile of the patient of less than one.

53. The method of claim 46, further comprising comparing the levels of the marker protein or fragments of the marker protein with a standard level, the standard level being an average of the levels of the marker protein or fragments of the marker protein in macrophages obtained from a plurality of individuals without the condition, wherein the levels of the marker protein or fragments of the marker protein are abnormal if there is a statistically significant difference from the standard level.

54. The method of claim 46, wherein the disease condition is Parkinson's Disease, Multiple Sclerosis, FrontoTemporal Dementia, or Amyotrophic Lateral Sclerosis.

55. The method of claim 46, wherein the marker protein is ubiquitin, myelin basic protein, tau protein, or alpha-synuclein.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0064] FIG. 1 is a flowplot of monocytes from the CSF of a patient diagnosed with non-AD/MCI (top panel) and a corresponding flowplot from a blood donor (bottom panel). The encircled cell population is activated macrophages/microglia.

[0065] FIG. 2 is a graph showing the relative distribution of activated and non-activated macrophages from patients with 1: Alzheimer disease (AD), 2: Multiple sclerosis (MS), 3: no CNS disease, 4: MCI/non-AD and 5: 7-10 days post stroke.

[0066] FIG. 3 shows IP-MS spectra of Aβ peptide fragments from CSF samples from patients. The two upper spectra are from AD patients, N=10 (F=8), Mean age=68.9, #CDI6+˜1894 cells. The two lower spectra are from. MS patients, N=3 (F=2), Mean age =45, #CD16+˜773 cells.

[0067] FIG. 4 shows IP-MS spectra of AP peptide fragments from CSF samples from individuals. The spectra are from individuals with no CNS disease, N=13 (F=8), Mean age=36.15, #CD16+˜4792 cells.

[0068] FIG. 5 shows IP-MS spectra of AP peptide fragments from CSF samples from patients. The two upper spectra are from patients with MCI but without AD, N=5 (F=3), Mean age =71.4, #CD 16+˜1082 cells. The two lower spectra are from patients 7-10 days post stroke, N=5 (F=0), Mean age=67, #CD16+CSF˜2930, #CD 16+blood˜3748 cells.

[0069] FIG. 6 shows IP-MS mass spectra of C- and N-terminally truncated Aβ peptides immunoprecipitated from CSF.

[0070] FIG. 7 shows IP-MS spectra of AP peptide fragments from peripheral blood samples from 7-10 days post stroke patient (CD 16.sup.++ upper spectrum and CD16 lower spectrum).

[0071] FIGS. 8a-8f are-schematic diagrams of a kit in accordance with one embodiment of the present invention, in use.

[0072] FIG. 9 is a flow diagram showing the steps in the production of antibodies for use in a kit in accordance with one embodiment of the present invention.

[0073] FIGS. 10a-10b shows confocal microscopy images of a macrophage from CSF stained for intracellular MBP, where FIG. 10a is a fluorescent image of the cell and

[0074] FIG. 10b is a transilluminated image of the same cell.

[0075] FIGS. 11a-11b shows confocal microscopy images of a macrophage from CSF stained with isotype-specific antibodies, where FIG. 11a is a fluorescent image of the cell and FIG. 11b is a transilluminated image of the same cell.

[0076] FIGS. 12a-12b shows confocal microscopy images of a macrophage from blood stained with intracellular MBP, where FIG. 12a is a fluorescent image of the cell and FIG. 12b is a transilluminated image of the same cell.

[0077] FIGS. 13a-13b shows confocal microscopy images of a macrophage from blood stained with isotype-specific antibody, where FIG. 13a is a fluorescent image of the cell and FIG. 13b is the transilluminated image of the same cell.

[0078] FIGS. 14a-14b shows confocal microscopy images of a macrophage from blood with no primary antibody against intracellular antigens, where FIG. 14a is a fluorescent image of the cell and FIG. 14b is a transilluminated image of the same cell.

[0079] FIGS. 15a-15b shows graphs of Means Fluorescent Intensity in CSF 15a and Blood 15 b against cell count.

BRIEF DESCRIPTION OF THE SEQUENCES

[0080] SEQ. ID NO. 1 is the amino acid sequence of the Aβ protein.

[0081] SEQ. ID NO. 2 is the amino acid sequence of the Ubiquitin protein (RPS27A)

[0082] SEQ. ID NO. 3 is the amino acid sequence of the Myelin basic protein (MBP).

[0083] SEQ. ID NO. 4 is the amino acid sequence of the Tau protein (MAPT).

[0084] SEQ. ID NO. 5 is the amino acid sequence of the alpha-synuclein protein (SNCA).

DETAILED DESCRIPTION

[0085] In embodiments of the present invention, the peptide/protein content of activated macrophages is used for the early diagnosis of AD as will now be explained. The data disclosed herein (see, for example, FIGS. 3 to 5) indicate that macrophages in AD have a reduced capacity for Aβ phagocytosis. Most of the patients studied as reported herein are patients with AD according to Dubois et al.[12], with MCI but not dementia. This suggests that reduced phagocytosis may be present early in disease development.

[0086] An embodiment of the present invention will now be described. A sample of cerebro-spinal fluid (CSF) is obtained from a patient by lumbar puncture. The macrophages in the sample are stained with fluorescent-labelled anti-CD14 and anti-CD16 antibodies and the macrophages are then withdrawn from the sample by fluorescence activated cell sorting.

[0087] The cells are selected on the basis of CD14 and CD16 expression because this enables activated macrophages to be differentiated from quiescent cells (increasing CD16 expression signifies an activated status). This technique also avoids the inadvertent sampling of other cell types such as CD68 positive dendritic cells. This approach contrasts with that reported by Fiala et al (39) in which CD68 positive cells were selected.

[0088] The resultant macrophage cells are lysed and prepared for protein analysis. The cell lysate is mixed with monoclonal antibodies capable of binding fragments of the Aβ protein (SEQ. ID NO.: 1). Exemplary antibodies are 6E10, 4G8 and 11A50-B10 from Signet Laboratories, Inc. The 6E10 antibody is used to immunoprecipitate Ap fragments 1-16, the 4G8 antibody immunoprecipitates AP fragments 17-24 and the 11A50-B10 antibody immunoprecipitates Ap fragments of 1-40. In alternative embodiments, a different panel of antibodies, specific for other fragments, may be used. The monoclonal antibodies are also coupled to magnetic beads, for example, with beads bound to anti-IgG antibodies. The magnetic beads are used to extract the fragments of the Aβ protein. The antibodies and beads are subsequently removed from the peptide fragments. The peptide fragments are then analysed by MALDI-TOF mass spectrometry and the sequence of the fragments derived from the molecular mass of each fragment. The results are displayed quantitatively to indicate the relative quantity of each fragment. Where no Aβ protein or Aβ protein fragments are detected in the macrophages, this is indicative that the patient has AD.

[0089] The Aβ fragments shown in the IP-MS spectra result from intracellular degradation according to the character of the catalytic active site and conditions of action of intracellular protease/peptidases. Such fragments do not necessarily correspond to the sequences of Aβ fragments found extracellularly in CSF. Thus in some embodiments, in order to identify the exact length of each Aβ fragment obtained in the experiment, the peptides are isolated for determination of their respective amino acid sequences.

[0090] It is to be appreciated that the method described above detects the presence of the Aβ protein fragments that are present in vivo in the patient. The method does not involve a separate step of exposing the macrophages to the Aβ protein, in vitro, after extraction from the patient.

[0091] In some embodiments, the level of the Aβ protein fragments detected is compared with the level detected in a control individual who does not have AD. In such embodiments, a comparison is made between the level and pattern of Aβ protein fragments from the patient and those of the control individual. Where the level of Aβ protein fragments is significantly below that in the control individual then this is indicative of AD in the patient. Similarly, if the type of Aβ protein fragments present in the individual is significantly different from those in the control individual then this is indicative of AD in the patient. In alternative embodiments, a standard level of Aβ protein fragments is generated by detecting the presence of such fragments in the macrophages in CSF in a plurality of control individuals who do not have AD. The level and pattern of Aβ protein fragments from the patient is then compared with the standard level and a statistically significant reduction in level or difference in pattern of the presence of fragments is indicative of AD.

[0092] In other embodiments, a single patient is examined annually over a period of time (e.g. 10 years). On each occasion, the levels of Aβ protein fragments in the macrophages in a CSF sample from the patient is studied as described above. A significant change in the level or pattern of Aβ protein fragments each year, in particular a reduction in the level of the Aβ protein fragments year on year, is indicative of the presence of AD.

[0093] In certain embodiments, additional AD markers in the patient are also measured, at the same time as the above-described analysis is carried out. Such additional AD markers include abnormal levels of Aβ42, Tau, Phospho-Tau or the Aβ42/Aβ40 ratio in a CSF sample obtained from the patient or in the RNA profile of a blood or CSF sample obtained from the patient. An abnormal level of some or all of these additional AD markers as well as an abnormal level of Aβ protein fragments in the macrophages in the CSF of the patient is indicative of the presence of AD. An exemplary abnormal (i.e. pathological) level of Aβ42 is a CSF concentration of less than 550pg/ml. The concentration of Tau is age-dependent, high levels being pathological. A pathological level of Phospho-Tau is a CSF concentration of greater than 85pg/ml. A pathological level of the Aβ42/Aβ40 ratio is where (Aβ42/Aβ40)×10 is less than 1.

[0094] In the above described embodiments, a CSF sample from the patient is obtained. However, in alternative embodiments, a different type of sample is studied, for example, a blood sample. Such an alternative sample may be used because macrophages circulate from the bone marrow to the CNS and therefore macrophages in the blood of a patient may have been exposed to proteins in the CNS. It is, of course, easier to obtain a blood sample than a CSF sample from a patient.

[0095] One embodiment of the present invention uses the following criteria as the basis of a diagnostic test to assess Alzheimer's disease in a patient's activated macrophages/microglia: 1) Fulfilment of disease criteria, 2) Presence and sorting of CD16+ population of cells in CSF and blood with flow cytometry, 3) Presence /absence of Aβ peptide fragments in MS spectra after immunoprecipitation with antibodies, 4) Tailored methods at clinics. Flow cytometry and IP-MS can be replaced by other methods for sorting or distinguishing of cell subtypes and peptide fragment analysis.

[0096] Methods of Evaluating Fulfillment of Disease Criteria

[0097] The patient undergoes a thorough clinical investigation, including a study of medical history, physical, neurological and psychiatric examination, screening laboratory tests and MRI and PET imaging of the brain. The diagnosis of AD is made according to recently published criteria [12]. The patient undergoes a thorough physical and psychological examination when enrolled in the diagnosis programme at a hospital. The examination includes neuropsychological questionnaires for identification of cognitive deficits, neurological examination, genetic analysis, CSF biomarkers, imaging and metabolic profile.

[0098] Methods of Evaluating Presence and Sorting of CD16+ Population of Cells in CSF and Blood with Flow Cytometry

[0099] Cells are acquired on a FACSAria Cell-Sorting System and analysed using FACSDiva software (both Becton Dickinson). CSF cell populations are sorted based on their expression of relevant surface markers (CDs). Cells are gated according to forward- and side light-scattering properties and are positively selected for the presence of CD45+CD3+CD4+CD8 (characterisation of T-cell population), and CD45 +CD14 +CD16+CD19 (characterisation of activated macrophages and B-cell population). In order to preserve the immune cells intact, the cell sorting is performed at a maximum of four hours post puncture. CD14.sup.+/CD16.sup.+ sorted cells are lysed and kept frozen at −80° C. for further analysis (protein-analysis). In addition to collecting cells for protein analyses, the flow cytometry results indicate the CSF and periphery (blood) immune cell distribution for the patient.

[0100] Method of Preparation of Cells for Immunoprecipitation

[0101] CSF cell populations were sorted based on their expression of relevant surface markers (CDs). Cells were gated according to forward- and side light-scattering properties and were positively selected for the presence of CD45+CD3+CD4+CD8 (characterisation of T-cell population), and CD45+CD14+CD16+CD19 (characterisation of activated macrophages and B-cell population). Cell population and number of cells within each population were obtained and registered (see FIG. 2). The number of activated cells in 7-10 days post stroke patients is high, suggesting circulation of recruited cells also to the CSF compartment of a large number of immune cells. The number of activated macrophages in AD largely equals that in the MCI/non-AD group. The total of %-activated cells in MS is lower that AD; which may be because the immune process in MS mainly involves T-cells.

[0102] The sample CD14.sup.+/CD16.sup.+ and CD14.sup.+/CD16.sup.− sorted cells were washed with 400 μl PBS and centrifuged (4° C., 750×g, 5 min). The supernatant was removed and prepared for IP-MS analysis by adding 10 μL RIPA-buffer for cell lysis and keeping frozen at −80° C. prior to protein-analysis.

[0103] Method of Immunoprecipitation

[0104] An aliquot (4 μg) of the monoclonal antibodies 6E10 (1 mg/mL, epitope 4-9), 4G8 (1 mg/mL, epitope 18-22), or 11A50-B10 (0.5 mg/mL, reactive to the C-terminus) (Signet Laboratories, Inc.) was separately added to 50 μL magnetic Dynabeads (Sheep anti mouse, IgG) and incubated overnight on a rocking platform at +4° C. The remaining unbound antibody was removed by washing twice with phosphate-buffered saline (PBS, pH 7.4). After adding 1 mL CSF to the antibody-coated beads, the incubation was continued for an additional 1 h at +4° C. The beads were pelleted for 5 min by using a magnetic particle concentrator (Dynal MPC) and washed twice with PBS (pH 7.4) and twice with 50 mM ammonium bicarbonate (pH 7.3). After the final wash, the extracted Aβ peptides were eluted by adding 20 μL 0.5% formic acid (FA) in water. After vortexing for 2 min in room temperature, the beads were pelleted using the magnetic particle concentrator and the supernatant was collected. The collected supernatant was dried down in a vacuum centrifuge and redissolved in 5 μL 0.1% FA in 20% acetonitrile (ACN). All solvents used were of HPLC quality and all aqueous solutions were made using 18.2 M deionized water obtained from a Millipore purification system.

[0105] Methods of Evaluating Presence/Absence of Aβ in MS Spectra after Immunoprecipitation

[0106] IP-MS is used to isolate and determine the Aβ peptide content (Aβ signature) in the CD14.sup.+/CD16.sup.+ macrophages sorted by flow cytometry. Proteolytically processed Aβ peptides are difficult to detect using standard proteomic methods possibly because they comprise a heterogeneous set of both N- and C-terminally truncated peptides, some at low quantity. IP-MS analysis has been used previously to obtain an Aβ peptide signature successfully [43] [44](see FIG. 6). Briefly, the AP peptides are isolated from lysed macrophages using anti Aβ monoclonal antibodies and magnetic Dynabeads. Then a matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) analysis is performed on the immunoprecipitated peptides and the macrophage Aβ signature is calculated. The Absence of Aβ signal in the specimen is interpreted as a positive AD diagnosis.

[0107] Alternative Methodologies

[0108] In variants of the above-described methodology, the following techniques are used, [0109] 1. Instead of using flow cytometry to sort cells, activated macrophages/microglia cells are withdrawn using magnetic extraction, flotation techniques, or other antibody or affinity-based extraction techniques e.g. chromatography, gradient centrifugation, Alternatively the cells are studied using immunohistochemistry [0110] 2. Immune precipitation using other antibodies specific for the peptide/protein of interest. [0111] 3. Instead of using mass spectrometry, another technique for quantitative or semi-quantitative peptide/protein analysis is employed such as: HPLC-fluorescence or -UV, luminescence, streptavidin/biotin systems, immunohistochemistry a.o.

[0112] Alternative Conditions

[0113] In alternative embodiments a different pathological condition characterised by the presence of fragments of a marker protein in the brains of patients is studied. In each case it is necessary to identify the condition to he studied and the corresponding protein that characterises the condition. Exemplary conditions include: Parkinson's Disease in which ubiquitin (SEQ. ID NO: 2) is the characterising protein; Multiple Sclerosis where myelin basic protein (SEQ. ID NO: 3) characterises the condition; FrontoTemporal Dementia and Amyotrophic Lateral Sclerosis which are characterised by the tau protein (SEQ. ID NO: 4); and Parkinson's Disease (SEQ. ID NO: 5), Lewy body dementia and AD which are characterised by the alpha-synuclein protein. In each case, the method of detection or monitoring is carried out as is described above in relation to AD except that the antibodies used to immunoprecipitate the peptides from the macrophages are substituted with antibodies that are capable of binding fragments of the characterising protein of the condition, Furthermore, in the case of Multiple Sclerosis, abnormally high levels of the ubiquitin marker protein are indicative of the presence of the condition.

[0114] In some embodiments, multiple such conditions are tested for simultaneously by immunoprecipitating cell lysates with multiple sets of antibodies, each set of antibodies being specific for fragments of different characterising proteins.

[0115] In some embodiments of the invention, a diagnostic kit is provided in order to enable the detection of a pathological condition of the invention (that is to say a condition characterized by the presence of fragments of a marker protein in the brain of a patient suffering from the condition). The kit is suitable for use in ordinary clinical laboratories since it is based on an ELISA/immuno-PCR technique and so does not require the use of MALDI-TOF or IP-MS techniques as described in some previous embodiments. The kit comprises a panel of target specific antibodies which are specific for a first epitope of the marker protein. Thus, for example, where the pathological condition to be detected is Alzheimer's disease, the marker protein is the Abeta 42 protein. The kit also comprises a supply of magnetic heads which display macrophage specific antibodies (for example, antibodies specific for the CD14 and CD16 cell markers); a cell lysing agent such as RadioImmuno Precipitation Assay (RIPA) Buffer containing 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% Sodium deoxycholate and 0.1% SDS (Pierce Biotechnology); and a secondary antibody which is specific for a second epitope of the marker protein. The secondary antibody is conjugated to a double-stranded DNA marker molecule.

[0116] Referring to FIG. 8, the kit will now be described in use. A sample, such as a peripheral blood or CSF sample, is obtained from a patient and macrophage cells are isolated from the sample by mixing with the magnetic beads provided in the kit. The macrophage specific antibodies displayed by the magnetic beads bind the macrophages in the patient sample and the macrophages and the magnetic beads are then removed from the sample by magnetic means. The macrophage cells are then released from the macrophage specific antibodies by adjusting the pH of the solution and the macrophage cells 1 are lysed with the lysing agent in order to release the cell contents 2 which includes the marker protein 3 as shown in FIG. 8A.

[0117] Also provided in the diagnostic kit is a solid support 4 on which are immobilised a plurality of target antibodies 5, 6 which are specific for the marker protein 3.

[0118] As shown in FIG. 8b, the contents 2, 3 of the lysed macrophage cell 1 are then contacted with the solid support 4 such that the first epitope of the marker protein 3 binds to the target antibody 5.

[0119] Referring to FIG. 8c, the solid support 4 is contacted with a secondary antibody 7 which is conjugated to a double-stranded DNA marker molecule 8, The secondary antibody 7 is specific for the second epitope of the marker protein 3 such that the secondary antibody 2 is immobilised on the solid support 4 where the marker protein 3 is present.

[0120] Unbound proteins and unbound secondary antibody are then washed out and removed (see FIG. 8d).

[0121] Referring to FIG. 8e, the washed solid support 4 is then subjected to real time PCR which melts the double stranded DNA marker molecule 8 and amplifies the copy number (see FIG. 8f) in order to identify the number of copies of the DNA marker molecule. The number of copies of the DNA marker molecule 8 after a predetermined number of cycles of PCR amplification is indicative of the starting number of DNA molecules. Furthermore, there is a one-to-one relationship between the starting number of DNA molecules and the number of bound marker proteins. Therefore, this immuno-PCR technique provides an accurate indication of the number of marker protein molecules in the patient sample.

[0122] Accordingly, such diagnostic kits allow a simple immunological method to be used in standard clinical laboratories which are available in all hospitals, private clinics and commercial laboratories in order to analyse patient samples in accordance with the present invention. The use of the kit of the invention does not require the use of expensive or advance laboratory instruments and detection using an immuno-PCR technique ensures high sensitivity.

[0123] The target antibodies of the diagnostic kit may be antibodies already known in the art which are capable of binding the marker protein, such as the antibodies 6E10, 4G8 and 11A50-B10 described above. however, further antibodies can be identified as is explained in the flow chart in FIG. 9 in relation to antibodies to the Abeta protein. The process begins with obtaining a suitable patient sample by extracting a cerebral spinal fluid sample by a lumbar puncture (box 9). Subsequently, the activated macrophages from the sample are extracted by binding of labelled antibodies to suitable cell markers (e.g. CD14 and CD16) in the patient sample and sorting of the cells by FACS (see box 10). The isolated macrophages are then cultivated (box 11) and are encouraged to phagocytosis by challenging with the Abeta protein in vitro (box 12). Further details of this step are provided in the “amyloid-beta stress test” of Fiala et al (39). The cultivated macrophages are then lysed and digested fragments of the Abeta protein are extracted and immunoprecipitated (box 13).

[0124] At this stage in the procedure, the Abeta fragments may, themselves, be used to immunise host animals (such as rabbits) in order to produce antibodies specific for the Abeta protein fragments on a small-scale (box 14). However, for larger scale production of antibodies, the procedure continues with the identification of the digested Abeta protein fragments using tandem mass spectrometry (c,g. electrospray-Q-TOF or MALI-TOP (box 15)). Once the sequences of the Abeta protein fragments have been identified, the fragments are synthesised, for example by recombinant expression in a host cell such as E. coli (box 16). The synthesised Abeta protein fragments are then used to immunise host animals (usually rabbits) (box 17) which in turn produce antibodies (box 18). Antibodies may be obtained from the host animals and included in the diagnostic kits, but preferably, monoclonal antibody-producing cells are produced, as is known in the art (e.g. by production of hybridoma cells). Alternatively, the antibodies, or at least their complementarity determining regions, are sequenced and recombinantly expressed. Whichever method of antibody production is selected, the antibodies are purified and included in the diagnostic kit (box 19).

[0125] In some variants of the above-described diagnostic kits, a plurality of panels of antibodies are provided in the kit. For example, in one variant, the kit comprises first target antibodies that are specific for a first epitope of the Abets, protein and second target antibodies which are specific for a third epitope of the Abeta protein. In still further embodiments, a plurality of panels of antibodies are provided in the kit and the antibodies are specific for marker proteins corresponding to more than one pathological condition. For example, in one particular variant, a panel of antibodies is provided which is specific for the Abeta protein (the marker protein for Alzheimer's disease) and a panel of antibodies is provided specific for Multiple Sclerosis (where myelin basic protein is the marker protein). In these variants, it is preferred that different panels of secondary antibodies, each specific for a respective marker protein and each conjugated to a different DNA marker molecule, are provided such that the signal for the detection of each marker protein is distinguishable.

[0126] In the above described embodiments of the diagnostic kit, the detectable label is a DNA marker molecule. However, in other embodiments, a different detectable label is used. For example, the detectable label may be a fluorophore, a latex microbead or a gold particle. Such alternative detectable labels arc useful when the kit is provided only to provide a qualitative result rather than a quantitative result.

[0127] In some alternative embodiments of the kit, a lysing agent, as such, is not provided. Instead, cells are lysed mechanically, e.g. by centrifugation, prior to isolation of the macrophages.

[0128] It is also to be appreciated that the diagnostic kits of the present invention are not limited to kits comprising antibodies. In alternative embodiments, the antibodies of the kit are replaced with other binding reagents such as antigen binding fragments (e.g. F(ab′).sub.2 fragments or Fab fragments) or a polynucleotide sequence. Typically such other binding reagents have binding affinities for their target comparable to that of antibodies such as having a binding affinity of less than 100 nm in an aqueous buffered solution at between pH 4 and 8.

EXAMPLES

Example 1

Patient Selection

[0129] Patients were ambulatory or intramural and were recruited from Nevroklinikken at Akershus University Hospital Lumbar puncture was performed as a planned procedure. The patient groups were divided into following diagnosis: 1) probable Alzheimer's disease (AD) diagnosed according to NINCS-ADRA criteria [12]; 2) probable Multiple sclerosis (MS) diagnosed according to the McDonald criteria; 3) no nervous system disease (e.g. ME, and other patients with a full negative investigation for “organic” disease); 4) mild cognitive impairment (MCI)/non-AD; and 5) 7-10 days post-stroke patients.

Example 2

Lumbar Puncture/Blood Sampling

[0130] The lumbar puncture was routinely carried out in connection with diagnosis between 0900 and 1330 hrs. CSF MIS obtained from patients through lumbar puncture between vertebras L4 and L5 with the patients in horizontal positions. The skin in the lumbar region was thoroughly washed with sterile cotton swabs and chlorhexidine 5%. The neurologist on call performed the lumbar puncture. Fine disposable needles were used (Becton Dickinson 20GA 3.5 IN 0.9×90 mm). The sample for flow cytometry analysis was collected as the final sample, altogether 2 mL (˜40 droplets) of CSF. The blood sample (EDTA or heparin) was taken immediately prior to or following the lumbar puncture.

[0131] Cells were acquired on a FACSAria Cell-Sorting System and analysed using FACSDiva software (both Becton Dickinson) within a maximum of four hours post puncture/blood sampling.

Example 3

Preparation and Analysis of CSF/Blood Samples by Flow Cytometry

[0132] 2 mL CSF and/or 4 mL blood was pelleted (4° C., 400×g, 10 min). The supernatant was removed and the remaining cell pellets were washed once with staining buffer (Becton Dickinson, San Jose, Calif.). The cell pellets were diluted in 1-2 mL of staining buffer and were centrifuged at 4° C., 400×g, 10 min. The supernatant was removed, and the sample was transferred to a flow tube. The sample was stained with a panel of fluorescent-labelled antibodies (2.5 μL CD4-FITC and CD19-FITC, 2.0 μL of CD8-PE, CD16-PE and CD45-PerCP, 1.5 μL CD3-APC and CD14-APC all from Becton Dickinson). The samples were incubated in a refrigerator for 15-20 minutes before adding 3-4 droplets of FACSFlow solution, mixed and made ready for flow cytometry analysis.

Example 4

Flow Cytometry Analysis and Cell Sorting

[0133] CSF cell populations were sorted based on their expression of relevant surface markers (CDs). Cells were gated according to forward- and side light-scattering properties and were positively selected for the presence of CD45+CD3+CD4+CD8 (characterisation of T-cell population), and CD45+CD14+CD16+CD19 (characterisation of activated macrophages and B-cell population). Cell population and number of cells within each population were obtained and registered (see FIG. 2). The number of activated cells in 7-10 days post stroke patients is high, suggesting circulation of recruited cells also to the CSF compartment a large number of immune cells. The number of activated macrophages in AD largely equals that in the MCI/non-AD group. The total of %-activated cells in MS is lower that AD; which may be because the immune process in MS mainly involves T-cells.

[0134] The sample CD14.sup.+/CD16.sup.+and CD14.sup.+/ CD16.sup.+ sorted cells were washed with 400 μl PBS and centrifuged (4° C., 750×g, 5 min). The supernatant was removed and prepared for IP-MS analysis by adding 10 μL RIPA-buffer for cell lysis and keeping frozen at −80° C. prior to protein-analysis.

Example 5

Pooling of Patients According to Diagnosis

[0135] The sorted cells were pooled together according to diagnosis, prior to IP-MALDI-TOF-MS analysis, in the following groups. [0136] 1) Alzheimer patients: N=10 (F=8), Mean age=68,9, #CD16+˜1894 cells [0137] 2) MS patients: N=3 (F=2), Mean age-.sup.-- 45, #CD16+˜773 cells [0138] 3) No CNS disease, N=13 (F=8), Mean age=36,15, # CDI6+˜4792 cells [0139] 4) MC1/non-Alzheimer: N=5 (E=3), Mean age =71,4, # CD16˜1082 cells [0140] 5) 7-10 days post stroke N=5 (F=0), Mean age =67, # CD 16+CSF˜2930, #CD16+blood˜3748 cells

Example 6

Immunoprecipitation-Matrix-Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry (IP-MALDI-TOF-MS)

[0141] Samples were immunoprecipitated as described above. MALDI samples were prepared with the seed layer method. Briefly, a seed layer was created on a MALDI-TOF MS stainless steel sample probe (Bruker Daltonics Inc.) by depositing 0.5 μL (1 g/L) of alfa-cyano-4-cinnamic acid (CHCA, Fluka) dissolved in ACN. One microliter of saturated (15 g/L) CHCA in 0.1% trifluoroacetic acid in ACN/water (1:1 v/v) was added to an equal volume of the dissolved peptides and mixed. One microliter matrix/peptide solution was added to the probe and the sample was left to dry completely in air. MALDI-TOF MS measurements were performed using an AUTOFLEX instrument (Broker Daltonics Inc.) operating in reflecting mode at 19 kV acceleration voltage. The spectra represent an average of 900 shots and were recorded up to 4600 Da. The spectra were calibrated using internal calibration (m/z 1826.8, 2068.0, 4130.0, and 4328.2) and each sample was analyzed in duplicate. All mass spectra were analyzed using Broker Daltonics flexanalysis 2.4, baseline subtracted and then smoothed with a 5-point Savitsky-Golay smooth. The results are shown in FIGS. 3 to 5, with two mass spectra per sample.

[0142] The results using IP-MS which are reported herein show that Aβ peptide fragments can be measured in activated macrophage/microglia subgroups, and differences in Aβ peptide content are related to disease type even though activated macrophage/microglia cells are present in normal numbers. Furthermore, the results show Aβ peptide fragments in macrophages/microglia in all control- and patient groups (the MS group, no CNS disease group and 7-10 days post stroke group) except AD patients, which show no detectable signal in the Aβ mass spectra (FIGS. 3 to 5).

[0143] These results suggest that despite having a distributed serious brain disease, the macrophage/microglia system in AD patients is not significantly activated compared with patients with no organic brain disease and post-stroke patients (compare groups 1, 3 and 5 in FIGS. 2 to 5). Furthermore, in contrast to all other patient groups, activated AD macrophage/microglia cells do not contain Aβ peptide fragments which indicates that their ability to phagocytose Aβ peptides is impaired or absent altogether.

[0144] The Aβ peptide content of macrophages in non-AD individuals does not correspond to the normal Aβ protein degradation pattern in CSF that arises from excision of APP by amyloidogenic proteases, β- and γ-secretase (see FIG. 6). The different degradation patterns seen in macrophages from CSF and blood (see FIGS. 3 to 5) may result from proteolysis of Aβ protein within the macrophages. The results disclosed herein are based on 62 patients (see FIG. 2).

Example 7

Ultrastructure (Filtrating/SEM)

[0145] Scanning electron microscope (SEM) analysis of CSF CD14+/CD16+ sorted macrophages is used as a complement to flow cytometry, IP-MS and other techniques, in order to obtain an overall picture of morphology of activated macrophages versus non-activated macrophages. Putative infectious agents, other cells and debris in the CSF are visualized with this technique.

[0146] Untreated CSF, and the sorted cell solutions (selected according to CD14.sup.+/CD16.sup.+ and CD14.sup.+/CD16.sup.++ properties) containing activated and non-activated macrophages, are applied on to the surface of a polycarbonate 0.6 run filter (Nucleopore, Inc), fitted to an airtight gadget (Gislaved, Sweden), vacuum filtered and immediately coated with a 40 ∈ thick layer of ionised gold for SEM. SEM is performed using a Philips High Resolution SEM (515). The morphology of these cells implies an active phagocytic status.

Example 8

[0147] The procedures of Examples 1 to 4 and 6 were repeated with respect to peripheral blood samples obtained from a 7-10 days post stroke patient. However, there was no pooling of patients. IP-MS spectra were generated in order to detect the presence of A13 peptide fragments. The spectra are shown in FIG. 7.

[0148] The upper spectrum shows CD16.sup.++ cells from peripheral blood. The number of CD16.sup.++ blood cells was 137. The peaks shown in this spectrum appear identical to the results shown in FIG. 5, lower spectrum (although the sample is drawn from a different patient). The lower spectrum in FIG. 7 shows CD16.sup.− cells from peripheral blood from one patient without Aβ pathology. The number of CD16.sup.− blood cells was ˜35,000.

[0149] This example demonstrates that blood samples of a patient can be analysed in order to assess the phagocytosis of Aβ peptides by macrophages.

[0150] Example 9

Intracellular Staining of Macrophages from Patient with Probable Multiple Sclerosis with Anti Myelin Basic Protein (MBP) Antibodies

[0151] Approximately 4.5 mL cerebrospinal fluid and 4.5 mL peripheral blood from a patient with probable Multiple Sclerosis were pretreated according to Example 3 before staining the cells with CD14-APC surface antibodies. Thereafter the cells were fixed and permeabilized with a formaldehyde/saponin-based reagent (IntraPrep) from Beckman coulter. The cells from CSF and blood were divided into 3 aliquots each and stained intracellularly with two different anti-myelin basic protein (MBP) antibodies from Epitomics (UniProtID P02686) and from Sigma (corresponding to residues 102-116 of human MBP). A non-specific isotype specific antibody (Rabbit IgG) from Epitomics was used as a control. In addition a sample with only CD14-APC staining was included. A secondary antibody (goat anti-rabbit IgG) from Invitrogen, fluorescently labeled with AlexaFluor488, was used to detect binding of primary antibody to antigen.

[0152] Flow cytometry was performed as described in Example 4. Cells were sorted according to their CD14.sup.+/intracellular signal, spotted onto a glass slide coated with polysine that attracts and adheres to cells. ProLong Gold antifade reagent (Invitrogen) was added to the slides (a mounting medium with DAPI), which enhances resistance to photobleaching and gives an additional staining of nucleus. A Leica confocal microscope was used to visualize cells.

[0153] FIGS. 10a and b show a macrophage from CSF stained for intracellular MBP. Granular structures stained positive for MBP (white arrows).

[0154] FIGS. 11a and b show a macrophage from CSF stained with isotype specific antibody control.

[0155] FIGS. 12a and b show a macrophage from blood stained for intracellular MBP. Granular structures stained positive for MBP(white arrows).

[0156] FIGS. 13a and b show a macrophage from blood stained with isotype specific antibody control.

[0157] FIGS. 14a and b show a macrophage from blood with no primary antibody against intracellular antigens. The fluorescent signal is due to autofluorescense.

[0158] FIGS. 15a and b shows Mean Fluorescent Intensity (MFI) in CSF(a) and blood(b) from a patient with suspected MS. A MFI difference between the cells stained with MBP and cells stained with isotype specific antibody and a negative control (no primary antibody against intracellular antigens) is shown suggesting presence of bound anti-MBP giving an AlexaFluor 488 signal in the flowcytometer. Tabulated results are provided in Tables 1 (CSF) and 2 (peripheral blood).

TABLE-US-00001 TABLE 1 Number of Geometric CSF macrophages Mean FI Mean FI CV Rabbit Anti MBP 135 402 376 36.1 Rabbit Isotype control 104 328 292 98.9

TABLE-US-00002 TABLE 2 Number of Geometric PB macrophages Mean FI Mean FI CV Rabbit Anti MBP 767 1503 1419 32.07 Rabbit Isotype control 1036 783 766 19.8 Negative control 1340 169 163 30.7

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