A method for in vitro diagnosis of multiple sclerosis

Abstract

The invention concerns a method for in vitro diagnosis of multiple sclerosis (MS ) based on the absolute count of lymphocytes with the CD49d+CD154+ phenotype autoreactive toward myelin proteins after the exposure of Peripheral Blood Mononuclear Cells to myelin peptides in the in vitro conditions. The method is useful for the early diagnosis of multiple sclerosis.

Description

[0001] A method for in vitro diagnosis of multiple sclerosis

[0002] Description

[0003] Technical Field

[0004] The invention concerns a method for the diagnosis of multiple sclerosis (MS) (Latin: Sclerosis multiplex') based on the absolute count of lymphocytes with the CD49d+CD154+phenotype autoreactive toward myelin proteins when Peripheral Blood Mononuclear Cells (PBMC) are exposed to myelin peptides in the in vitro conditions. The method is useful for the early diagnosis of multiple sclerosis.

[0005] Background Art

[0006] MS is one of the most common immune system disorders in the global population. It may develop as one of the three primary types: relapsing-remitting, secondary progressive and primary progressive. It affects 2.8 million people worldwide, and the population of patients in Poland is estimated at approx. 45-50 thousand people (but it is underestimated) . MS is known as a disease of young adults, because diagnosis is typically established in individuals in an age range of 20-40 years [1]. Such high incidence is an important social issue, which involves the patient's elimination from social and job-related activity. One of the most important problems neurologists face is the lack of availability of early diagnosis, resulting from no diagnostic tests to confirm the medical condition at the early stage of its development or to identify an active process that results in the development of the disease.

[0007] The symptoms of the various types of MS are similar, but they differ between patients irrespective of their type of the disease. Most patients, both those with relapsingremitting and those with primary progressive types, experience increasing disability at a certain point in the course of the disease. An important objective of treating multiple sclerosis is to slow down the progression of disability as early as possible. The relapsing-remitting type of multiple sclerosis is typically identified at the age of 20-30 years. The primary progressive type of multiple sclerosis is typically identified 10 years later than the relapsing-remitting type and it may be diagnosed after the steady decrease in increasing disability has ceased and when other diseases have been ruled out. This means that diagnosis is typically established in individuals aged 40 and 50 years. The most recent progress in the research on MS has resulted in the development of therapeutic methods that can effectively slow down the progress of the disease provided that it is diagnosed early.

[0008] State of the art

[0009] Testing oligoclonal bands in cerebrospinal fluid is the only laboratory test reported in the literature, and it has never been accepted as a routine test. However, as it has not been validated and considering the complex and nonstandardized procedure involved (an isoelectric focusing technique on agarose gel in combination with the immunoblotting technique), ambiguity of interpretation (negative results at the early stage of the disease), the method has never been validated and applied in the routine diagnosis of MS. In addition, the sensitivity of the method of 80.4 % and specificity of 62.0 % was too low for the test to be useful for diagnosing the disease [1].

[0010] It is known from the state of the art that the subpopulation of lymphocytes with the CD49d+CD154+phenotype is specific for MS patients [2, 3]. The lymphocytes are vital in the process of impaired regeneration of the central nervous system (CNS). Tests have been performed to identify the subpopulation of cells that disrupts the maturation of oligodendrocyte precursors into oligodendrocytes that produce the myelin sheath and thus CNS regeneration after a flare of the disease [2, 3]. The authors have proved that the lymphocytes are found both in the peripheral blood of individuals with MS and in the animal model of MS in EAE (Experimental Autoimmune Encephalomyelitis) mice in the brain, thus confirming the validity of the theoretical assumption of the clinical relevance of the lymphocyte subpopulation [2, 3].

[0011] Summary of invention

[0012] No sensitive, specific and validated laboratory tests based on the analysis of peripheral blood are currently available to establish definitive diagnosis when MS is suspected. The objective of the invention was to develop a method for the diagnosis of multiple sclerosis based on a diagnostic test that monitors the emergence of CD49d+CD154+lymphocytes autoreactive toward myelin peptides in peripheral blood. The proposed solution is characterized in that it has high specificity, sensitivity and a relatively short time to obtain a result.

[0013] A method for in vitro diagnosis of multiple sclerosis (MS) of the invention includes the following steps:

[0014] Step 1: an isolation of a population of Peripheral Blood Mononuclear Cells (PBMC) from whole peripheral blood and culturing the PBMC cells with myelin peptides: PLP 178-191 (SEQ ID NO: 3), MOG 35-55 (SEQ ID NO: 1), MBP 86-99 (SEQ ID NO: 2) and anti-CD28 and anti-CD154PE antibodies in a culture medium, wherein as a first negative control, unstimulated PBMC cells in the culture medium are used, as a second negative control, unstimulated PBMC cells cultured with anti-CD28 and anti-CD154PE antibodies in the culture medium are used, and as a positive control, PBMC cells stimulated with S. aureus enterotoxin B together with anti-CD28 and anti-CD154PE antibodies in the culture medium are used;

[0015] Step 2: a labeling the PBMC cells with anti-CD154 and anti- CD49d antibodies and FVS780;

[0016] Step 3: a counting the PBMC cells with a double positive CD49d+CD154+phenotype labeled in Step 2;

[0017] Step 4: an interpretation of results, wherein the diagnosis of multiple sclerosis is established based on a resulting MS index calculated according to a formula: wherein the absolute value is defined as the count of autoreactive lymphocytes with respect to PLP 139-151 (SEQ ID NO: 3), MOG 35-55 (SEQ ID NO: 1), MBP 85-99 (SEQ ID NO: 2) proteins of the total count of all lymphocytes with the CD49d+CD154+phenotype, wherein the MS index of ≥1.3 is a positive result, namely suspected MS and / or development of the disease, the MS index of between >1.1 and <1.3 is an indeterminate result, and the MS index of ≤1.1 is a negative result, and wherein diagnosis is established on condition that a PC index, calculated according to a formula: is ≥1.3.

[0018] Preferably, the isolation of PBMC cells in Step 1 is performed using a density gradient centrifugation. Preferably, the density gradient centrifugation is performed at room temperature for 30 minutes, at the centrifuge speed of 400 x g, wherein after centrifugation a middle fraction containing PBMC cells is isolated.

[0019] Preferably, the concentration of each of the myelin peptides in Step 1 is 25 μg / mL.

[0020] Preferably, the counting the labeled PBMC cells with the double positive CD49d+CD154+phenotype in Step 3 is performed using flow cytometry.

[0021] Preferably, the counting in Step 3 is performed using a cytometer with a technical capability of determining a cell quantity in a predefined volume of the test sample, wherein the count value in the region of cells with the CD49d+CD154+phenotype is normalized to the predefined sample volume.

[0022] Alternatively, the counting in Step 3 is performed using a cytometer without a technical capability of determining a cell quantity in a predefined volume of the test sample, wherein the absolute value is calculated based on the count value of single cells in the region with the CD49d+CD154+phenotype and normalization to a specifically predefined quantity of microbeads.

[0023] Most preferably, the MS index is determined twice based on independent blood samples at an at least two-week interval.

[0024] The blood sample is preferably from a patient who has not been taking immunosuppressive medicines within two weeks before the test.

[0025] The method of the invention is based on a highly specific response of the immune system of MS patients to the antigen, that is, myelin sheath peptides: myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and proteolipid protein (PLP). The specific immune response in laboratory conditions may be monitored through the assessment of the expansion of the lymphocyte subpopulation with the CD49d+CD154+phenotype in the total population of Peripheral Blood Mononuclear Cells (PBMC), isolated from patients with suspected MS in the in vitro conditions. Based on the quantitative analysis of CD49d+CD154+lymphocytes autoreactive toward the myelin proteins it can be determined whether an immune response that may ultimately lead to the development of the disease is developing in the subject. This results from the fact that the emergence of autoreactive lymphocytes is a necessary and critical condition for the development of autoimmune diseases, such as MS. Earlier studies have proved that the phenotype of autoreactive CD49d+CD154+lymphocytes enables very good characterization of lymphocytes responsible for disrupting the myelination process of CNS neurons through the reprograming of oligodendrocyte precursors (cells responsible for myelin synthesis and formation of the myelin sheath around the nerve cell dendrites) [2, 3]. When stimulated ex vivo, the lymphocytes may be additionally induced to proliferate in the in vitro conditions through re-stimulation (another stimulation outside the human body) with peptides comprised in the myelin sheath that constitute the autoantigen in this medical condition (MBP, MOG and PLP). These properties have been used by the inventors to develop the method of the invention useful for the diagnosis of individuals with suspected MS.

[0026] Through the stimulation of cultured PBMCs with anti-CD28 and anti-CD154 (PE) antibodies, enhancement of immune response was achieved in the culture, so that the count of lymphocytes responding to the autoantigen in the in vitro conditions (MBP, MOG, PLP) is possible to detect. Owing to the co-stimulation (additional stimulation) with anti-CD28 and anti-CD154 antibodies, the quantity of cells with the CD49d+CD154+phenotype, among which autoreactive lymphocytes toward the myelin proteins were present, could be increased 10-15 times.

[0027] In the method of the invention, the absolute count of lymphocytes autoreactive toward myelin proteins was included in the total pool of all specific lymphocytes with the CD49d+CD154+phenotype, which is the normal physiological pool of cells associated with specific immune response to other antigens. To facilitate the interpretation of results, the MS index was used, which should be interpreted as the ratio of the quantity (absolute count) of CD49d+CD154+lymphocytes in the system after stimulation with myelin peptides (MBP / MOG / PLP) to the system of unstimulated cells.

[0028] Depending on the technical capabilities of the flow cytometer used, two variants of the estimation of the absolute count of the CD49d+CD154+lymphocyte subpopulation are possible: a) The cytometer has a technical capability of determining a cell quantity in a specific volume of the test sample (e.g. the Cytoflex LX cytometer from Beckman Coulter). In such a case, the count value in the region of cells with the CD49d+CD154+phenotype in each system (PBMCs stimulated / unstimulated with peptides) is normalized to a specific sample volume (e.g. to 500 μL). b) The cytometer does not have a technical capability of determining a cell quantity in a specific volume of the test sample (most of the commercially available devices, e.g. LSRII from Becton Dickinson). In such a case, the absolute value in both systems (PBMCs stimulated / unstimulated with myelin peptides) is calculated based on the count value of single cells in the region with the CD49d+CD154+phenotype and normalization to a specifically defined quantity of microbeads listed by the manufacturer.

[0029] Microbeads should be understood as synthetic microspheres with fluorescent properties and a strictly standardized size.

[0030] The method of the invention enables detection of single autoreactive CD49d+CD154+lymphocytes responding to myelin peptides among the approx. 20 thousand lymphocytes subjected to cytometry analysis. The method enables early identification of MS. Identification of the disease at the stage of developing specific immune response autoreactive toward myelin ensures additional time for a therapeutic intervention to slow down the progress of the disease or its alleviation before changes in the brain develop in the form of demyelination plaques resulting from the destruction of nerve cells by the autoreactive lymphocytes.

[0031] Brief Description of Drawings

[0032] The invention is illustrated by embodiments in the figure in which:

[0033] Fig. 1 shows the cytometry analysis of samples from a patient with MS (patient MS6). Figure 1A - cytometry analysis of labeled cells with the regions marked. Figure IB - statistical analysis of counts in the respective regions with the "hierarchy of regions". The MS index is shown at the bottom (test sample) with the index for positive control (PC index).

[0034] Fig. 2 shows the cytometry analysis of samples from a healthy individual (patient HC3). Figure 2A - cytometry analysis of labeled cells with the regions marked. Figure 2B - statistical analysis of counts in the respective regions with the "hierarchy of regions". The MS index is shown at the bottom (test sample) with the index for positive control (PC index).

[0035] Detailed description of the invention

[0036] Whole blood from the ulnar vein previously collected to a tube with an anticoagulant - heparin lithium (10 lU / mL) - is the starting material for conducting the method of the invention. Conducting of the method is divided into steps: Step 1 - isolation of PBMC cells from peripheral blood and setting up a PBMC cells culture; Step 2 - tricolor labeling of PBMC cells after the culture; Step 3 - cytometry analysis of labeled cells; Step 4 - interpretation and approval of results .

[0037] Examples

[0038] Methodology considerations for Step 1

[0039] Cell culture

[0040] The PBMC cells were obtained from whole blood from the ulnar vein (at least 10 mL, tubes containing 10 lU / mL heparin lithium) .

[0041] In the first step, the PBMC cells were isolated in the concentration gradient (e.g. Histopaque-1077, SIGMA). 10 mL of whole blood was applied on 10 mL of Histopaque-1077; this was subsequently centrifuged for 30 minutes in conditions of room temperature and centrifuge speed of 400 x g. After centrifugation, the middle fraction containing mononuclear cells was isolated. Subsequently, erythrocytes were lyzed for 6 min at room temperature (RT) (5 mL PharM Lyse Ammonium Chloride Lysing Reagent, Pharmingen cat. No. 555899).

[0042] In the subsequent step, erythrocyte lysis was stopped by adding 40 mL of the RPMI1640 medium (Sigma-Aldrich, cat. No. R8758) with the addition of 1 vol.-% penicillin (10,000 units) and streptomycin (10 mg) (Sigma-Aldrich cat. No. P4333) and 10 vol.-% FBS (Gibco, cat. No. 10082147) and centrifuged (10 min, 1400 rpm, RT). The resulting PBMC cells were suspended in 2 mL of the RPMI1640 medium (composition, see above) to calculate density (Btirker chamber), and approx. 8-15 million cells were obtained on average. The cells were brought to a density of 2 million / mL in the RPMI1640 medium with the addition of L-glutamine, penicillin and streptomycin, and FBS; and seeded onto a 96-well plate (U- bottom type) in the quantity of 200 μL of the RPMI1640 medium per well (cell density: 1 x 106per 1 mL) in the systems: a) Negative control 1 (first negative control): unstimulated PBMC cells + medium. The objective of negative control 1 was to set cut-off parameters (setting the region in which the appearance of cell signals would be considered a positive result). Control No. 1 and the setting of region 1 should be performed for each new batch of reagents. Negative control 1 may be applied once every several samples on condition that tests are performed on one day. b) Negative control 2 (second negative control): unstimulated PBMC cells + anti-CD28 antibodies (0.5 μL, BD Pharmingen cat. No. 555725) + anti-CD154PE antibodies (2.5 μL, Becton Dickinson and Company BD Biosciences, clone 89-76, cat. No. 648887) + medium. c) Positive control: PBMC cells stimulated with S. aureus enterotoxin B (SEB) (1 gg / mL, Sigma cat. No. S4881) + anti-CD28 antibodies (0.5 μL, BD Pharmingen cat. No. 555725) + anti-CD154(PE) antibodies (2.5 μL, Becton

[0043] Dickinson and Company BD Biosciences, clone 89-76, cat. No. 648887) + medium (RPMI1640 with the composition as specified above).

[0044] The objective of positive control was to rule out false negative results as a result of for example general immune system depletion or intake of immunosuppressive or anti-inflammatory medicines (steroids or cyclooxygenase inhibitors). SEB was used in the claimed invention because this was an antigen to which every subject developed specific immune response. d) Test sample: PBMC cells stimulated with myelin peptides:

[0045] PLP 139-151 (Eurogentec, catalog No. AS-62742, SEQ ID NO: 3), MOG 35-55 (Eurogentec, catalog No. AS-63918, SEQ ID NO: 1), MBP 85-99 (Eurogentec, catalog No. AS-62731, SEQ ID NO: 2), each peptide at a concentration of 25 gg / mL + anti-CD28 antibodies (0.5 mL, BD Pharmingen cat. No. 555725) + anti-CD154PE antibodies (2.5 μL,

[0046] Becton Dickinson and Company BD Biosciences, clone 89-76, cat. No. 648887) + medium (RPMI1640 with the composition as specified above).

[0047] The plated cells were cultured for 24 h in humid atmosphere at 37 °C, 5 % CO2.

[0048] Methodology considerations for Step 2

[0049] Tricolor labeling of cells with anti-CD154 and anti-CD49d antibodies and FVS780 (a dye to distinguish between dead and viable cells)

[0050] Depending on the type of the flow cytometer, labeling is performed in appropriate tubes: A) without microbeads, for cytometers having a technical capability of determining the quantity reading in a specific sample volume

[0051] B) with a specific constant quantity of microbeads, for cytometers not having a technical capability of determining the quantity reading in a specific sample volume.

[0052] In the demonstrative case hereby, a cytometer variant not having the technical capability of estimating the absolute level (count) of cells in a specific sample volume was used. Therefore, cell labeling was performed in cytometry tubes containing a constant quantity of microbeads as predefined value by the manufacturer (the quantity of microbeads in a tube is specified on the packaging with lot No. of the specific product batch). In this example, these were BD Trucount Tubes from Becton Dickinson and Company BD Biosciences (cat. No. 340334).

[0053] In the first step, to the tubes containing the quantity of microbeads specified by the manufacturer (tubes containing 49,500 in each tube were used) 310 μL of BBS stain buffer was added and, successively, CD154PE in the quantity of 10 μL per sample (Becton Dickinson and Company BD Biosciences, clone 89-76, cat. No. 648887), CD49dBB515 in the quantity of 5 μL per sample (BD Biosciences, clone 9F10, cat. No. 564593) and Fixable Viability Stain 780 (a dye to distinguish between dead and viable cells) in the quantity of 1 μL per sample (BD Biosciences, cat. No. 565388). Subsequently, 190 μL of cell suspension was added and it was incubated for 30 min at room temperature in the dark.

[0054] Methodology considerations for Step 3

[0055] Cytometry analysis Step 3 involved cytometry analysis of labeled cells (Fig. 1A) with the statistics of counts in the respective regions (Fig. IB, "hierarchy of regions"). The MS index is shown at the bottom of the figure (test sample) with the index for positive control (PC index). Fig. 1 shows the analysis of samples from a patient with MS (patient ID: MS6). Fig. 2 shows the analysis of samples from a healthy individual (patient ID: HC3). i) Flow cytometer settings (principal settings). cell acquisition parameters (Acquisition Controls): i) settings for regions saved for further analysis: all counts (Storage Gate: all events); ii) counts to be saved: all (Events To Record: all events); iii) region at which cell acquisition is stopped: 20,000 counts in the Pl region (Stopping Gate: Pl 20,000 events); iv) quantity of counts shown on the monitor during analysis: 1,000 counts (Events To Display: 1,000 events). focusing buffer flow parameters: low or medium (flow rate: low or medium) i) Settings for matrix regions

[0056] Area dedicated to CD49d+CD154+lymphocytes (P3 CD154 region vs. CD49d) is set for "negative control 1" so that there are no more than 25 cells per 20,000 of all acquired PBMCs in the area. The area dedicated to CD49d+CD154+lymphocytes is subordinate in the hierarchy to the region of all PBMCs (Pl region, see Fig. 1A and Fig. 2A, Step 3). • The area dedicated to microbeads (P5 region) is marked by referring labeled CD49d+CD154+lymphocytes in the tubes containing microbeads (e.g. BD Trucount Tubes) vs. labeled CD49d+CD154+cells in standard tubes used for flow cytometry (not containing microbeads) .

[0057] Methodology considerations for Step 4

[0058] Step 4 Interpretation and approval of results

[0059] Step 4 involved calculating CD49d+CD154+lymphocyte expansion index values and interpretation of results.

[0060] Deriving the calculation formula for the MS index in the variant with tubes with a specific quantity of microbeads, e.g. BD Trucount Tubes from Becton Dickinson

[0061]

[0062] The formula in parentheses may be omitted if all tubes have the same LOT number (identical quantities of microbeads in each tube).

[0063] If the tubes have different LOT numbers, the MS index needs to be calculated according to the following formula:

[0064] The quantity of microbeads for each tube is specified on the packaging of each product batch.

[0065] Total sample volume: 500 μL

[0066] Interpretation of results:

[0067] Calculation of the CD49d+CD154+lymphocyte expansion index after exposure to MBP / MOG / PLP (MS index) and the PC index after exposure to SEB in positive control test sample:

[0068] Wherein for flow cytometers having a technical capability of analyzing the reading quantity in a specific sample volume (calculation based on a normalized sample volume), the MS index is calculated from the formula:

[0069] For cytometers not having a technical capability of analyzing the reading quantity in a specific sample volume (calculation based on the normalized quantity of microbeads)

[0070] Interpretation of results:

[0071] Because the method of the invention is a functional test based on testing the reactivity of lymphocytes, certain conditions have been imposed as a recommendation rather than a necessary condition, to minimize the risk of producing a false negative or a false positive result.

[0072] The test is preferably performed twice based on independent blood collections in a two-week interval. The result for the patient is released when two tests have an identical interpretation value:

[0073] - MS index ≥1.3 - positive result (suspected MS and / or development of the disease);

[0074] - MS index in a range of between >1.1 and <1.3 - indeterminate result (known as gray zone);

[0075] - MS index ≤1.1 - negative result.

[0076] - When two results differ in terms of interpretation, the whole test should be performed again, preferably after one month. The test result may be affected by the overall health of the subject, intake of anti-inflammatory medicines, periodic hormonal changes, physical exercise. The period of 1 month enables minimization of the effect of the aforementioned factors on lymphocyte reactivity.

[0077] - When the test result is in the gray zone twice, it is indicated to monitor the patient every several months (2-4). The period corresponds to the dynamics of disease development from radiologically isolated syndrome (RIS) through clinically isolated syndrome (CIS) to the relapsing-remitting type of MS.

[0078] - PC index - positive control ≥1.3. The result can be approved.

[0079] - PC index - positive control <1.3. The test should be performed again based on a new blood collection with checking the validity of reagents and checking whether the test has been performed correctly.

[0080] - The patient should not take immunosuppressive medicines within 2 weeks before the test (immunosuppressive medicines suppress lymphocyte proliferation).

[0081] The usefulness of the proposed solution was proved based on six patients with confirmed disease (identified according to the 2010 McDonald criteria) and on 4 healthy volunteers. Table 1. Example analysis of six patients with confirmed disease - multiple sclerosis (MS), wherein the MS index value was above 1.3, and four healthy individuals (healthy controls, HC), wherein the MS index value was <1.1. The table lists values read from respective regions (P3 for

[0082] CD49d+CD154+, P5 for microbeads) and the value of the calculated MS index. All Trucount tubes were from the same batch and contained identical quantities of microbeads (49,500). The positive control result (PC index) in each test was >1.3.

[0083] As shown in Table 1, for patients with confirmed disease - multiple sclerosis, the MS index was >1.3 in each case, while the MS index in healthy individuals was each time below 1.1. An advantage of such solution is that the immune reaction is characterized by high antigenic specificity and, therefore, the proposed method is characterized by high specificity (a low rate of false positive results). Because quantitative analysis is performed on a flow cytometer (a device to analyze single cells), the proposed test is also characterized by high sensitivity (a low rate of false negative results).

[0084] LIST OF REFERENCES

[0085] 1. Calabrese M, Gasperini C, Tortorella C, Schiavi G, Frisullo G, Ragonese P, Fantozzi R, Prosperini L, Annovazzi P, Cordioli C, Di Filippo M, Ferraro D, Gajofatto A, Malucchi S, Lo Fermo S, De Luca G, Stromillo ML, Cocco E, Gallo A, Paolicelli D, Lanzillo R, Tomassini V, Pesci I, Rodegher ME, Solaro C; RIREMS group (Rising Italian Researchers in Multiple Sclerosis). "Better explanations" in multiple sclerosis diagnostic workup: A 3-year longitudinal study. Neurology. 2019 May 28; 92(22): e2527-e2537. doi: 10.1212 / WNL.0000000000007573.

[0086] 2. Piatek P, Namiecinska M, Domowicz M, Wieczorek M, Michlewska S, Matysiak M, Lewkowicz N, Tarkowski M, Lewkowicz P. Multiple Sclerosis CD49d+CD154+As Myelin- Specific Lymphocytes Induced During Remyelination. Cells. 2019 Dec 19; 9(1): 15. doi: 10.3390 / cells9010015.

[0087] 3. Piatek P, Namiecinska M, Domowicz M, Przygodzka P, Wieczorek M, Michlewska S, Lewkowicz N, Tarkowski M, Lewkowicz P. MS CD49d+CD154+Lymphocytes Reprogram Oligodendrocytes into Immune Reactive Cells Affecting CNS Regeneration. Cells. 2019 Nov 25; 8(12): 1508. doi: 10.3390 / cells8121508 .

[0088] SEQUENCE LISTING <110> MEDICAL UNIVERSITY OF LODZ <120> A method for in vitro diagnosis of multiple sclerosis <130> 58977 / 23 <160> 3 <170> BiSSAP 1.3.6 <210> 1 <211> 21 <212> PRT <213> Homo sapiens <220> <223> MOG (35-55) <400> 1

[0089] Met Glu Vai Gly Trp Tyr Arg Pro Pro Phe Ser Arg Vai Vai His Leu 1 5 10 15

[0090] Tyr Arg Asn Gly Lys 20 <210> 2 <211> 14 <212> PRT <213> Homo sapiens <220> <223> MBP (86-99) <400> 2

[0091] Vai Vai His Phe Phe Lys Asn lie Vai Thr Pro Arg Thr Pro 1 5 10 <210> 3 <211> 14 <212> PRT <213> Homo sapiens <220> <223> PLP (178-191) <400> 3

[0092] Asn Thr Trp Thr Thr Cys Gin Ser lie Ala Phe Pro Ser Lys 1 5 10

Claims

Claims1. A method for in vitro diagnosis of multiple sclerosis (MS) which includes the following steps:Step 1: an isolation of a population of Peripheral Blood Mononuclear Cells (PBMC) from whole peripheral blood and culturing the PBMC cells with myelin peptides: PLP 178-191 (SEQ ID NO: 3), MOG 35-55 (SEQ ID NO: 1), MBP 86-99 (SEQ ID NO: 2) and anti-CD28 and anti-CD154PE antibodies in a culture medium, wherein as a first negative control, unstimulated PBMC cells in the culture medium are used, as a second negative control, unstimulated PBMC cells cultured with anti-CD28 and anti-CD154PE antibodies in the culture medium are used, and as a positive control, PBMC cells stimulated with S. aureus enterotoxin B together with anti-CD28 and anti- CD154PE antibodies in the culture medium are used;Step 2: a labeling the PBMC cells with anti-CD154 and anti-CD49d antibodies and FVS780;Step 3: a counting the PBMC cells with a double positive CD49d+CD154+phenotype labeled in Step 2;Step 4: an interpretation of results, wherein the diagnosis of multiple sclerosis is established based on a resulting MS index calculated according to a formula:wherein the absolute value is defined as the count of autoreactive lymphocytes with respect to PLP 178-191 (SEQ ID NO: 3), MOG 35-55 (SEQ ID NO: 1), MBP 86-99 (SEQ ID NO: 2) proteins of the total count of all lymphocytes with the CD49d+CD154+phenotypewherein the MS index of ≥1.3 is a positive result, namely suspected MS and / or development of the disease, the MS index of between >1.1 and <1.3 is an indeterminate result, and the MS index of ≤1.1 is a negative result, and wherein diagnosis is established on condition that a PC index, calculated according to a formula:is ≥1.3.

2. The method of Claim 1, characterized in that the isolation of PBMC cells in Step 1 is performed using a density gradient centrifugation.

3. The method of Claim 2, characterized in that the density gradient centrifugation is performed at room temperature for 30 minutes, at the centrifuge speed of 400 x g, wherein after centrifugation a middle fraction containing PBMC cells is isolated.

4. The method of Claim 1, characterized in that the concentration of each of the myelin peptides in Step 1 is 25 μg / mL.

5. The method of Claim 1, characterized in that the counting the labeled PBMC cells with the double positive CD49d+CD154+phenotype in Step 3 is performed using flow cytometry.

6. The method of Claim 5, characterized in that the counting in Step 3 is performed using a cytometer with a technical capability of determining a cell quantity in a predefined volume of the test sample, wherein the count value inthe region of cells with the CD49d+CD154+phenotype is normalized to the predefined sample volume.

7. The method of Claim 5, characterized in that the counting in Step 3 is performed using a cytometer without a technical capability of determining a cell quantity in a predefined volume of the test sample, wherein the absolute value is calculated based on the count value of single cells in the region with the CD49d+CD154+phenotype and normalization to a specifically predefined quantity of microbeads.

8. The method of Claim 1, characterized in that the MS index is determined twice based on independent blood samples at an at least two-week interval.

9. The method of Claim 1, characterized in that the blood sample is from a patient who has not been taking immunosuppressive medicines within two weeks before the test.

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