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    • 专利摘要:
    Serum IgM antibody against phosphatidylcholine (IgM-PC) to be used as a biomarker of multiple sclerosis (MS) and of the different phases of this disease, especially the early CIS and EMRR phases, once experimentally verified using an ELISA technique adapted, a surprisingly high level of these antibodies in the corresponding stages of MS compared to controls, which triples the value of each cohort group, and doubles the level of serum IgM-LC antibodies from the same patients; and a new semiquantitative ELISA technique developed for the specific detection of said antibody in serum samples from potential MS patients, as a highly sensitive and accurate diagnostic method. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2768783A1
    申请号:ES202030053
    申请日:2020-01-23
    公开日:2020-07-02
    发明作者:Sádaba Maria Cruz；Morón Ursula Muñoz；Lirola Esther Escudero；Francisco J Quintana；Veit Johanne Rothhammer
    申请人:Fundacion Universitaria San Pablo CEU；
    IPC主号:
    专利说明:

    [0002] Serum IgM-PC antibody as a biomarker of multiple sclerosis, and a new ultrasensitive ELISA for the diagnosis of this disease
    [0004] The object of the present invention encompasses two aspects of special relevance in the differential diagnosis of multiple sclerosis (MS): On the one hand, the serum IgM antibody against phosphatidylcholine (IgM-PC) to be used as a biomarker of MS and of the different stages of the disease, especially the early stages CIS and RRMS; and, on the other, the semi-quantitative ELISA technique developed for the specific detection of said antibody in serum samples from potential MS patients, as a highly sensitive and precise diagnostic method.
    [0006] Known from various experimental studies that lipids, as the main component of the myelin layer of nerve fibers, are the main target of the autoimmune response in MS, and taking into account the role suggested by the data for IgG antibodies and IgM versus lipids as biomarkers in MS, and recent studies revealing that intrathecal production of IgG and IgM versus lactosylceramide (LC) and phosphatidylcholine (PC) is closely related to the neurodegeneration characteristic of MS, has been performed the quantification of IgG and IgM antibodies against said phospholipids in serum samples by an adapted ELISA technique, of high sensitivity, obtaining as a result a surprisingly high level of serum IgM-PC antibodies in patients with MS, which also reveal an association with the CIS, RR, SP and PP stages of the disease, especially for the first two, without detecting relevant differences for to the other proposed antibody.
    [0008] Based on this capacity of the serum antibody IgM-PC as a biomarker of MS, the ELISA technique applied for the detection of IgM-PC in serum samples from patients susceptible to MS has been standardized, with the sensitivity and precision demonstrated, according to a series of stages that include procedural parameters and resources that solve the problems inherent in immunochemical techniques for detecting antibodies in serum samples from patients with autoimmune diseases, and that make it a highly reliable diagnostic method for MS and subtypes.
    [0010] Therefore, IgM-PC is available to the pharmaceutical and hospital industry, and to the scientific community in general, as a new serum biomarker of MS and subtypes, and a new semiquantitative ELISA type diagnostic method for the detection of IgM-PC antibodies in blood, with high sensitivity and precision.
    [0012] SCOPE.-
    [0014] The present invention can be framed in two related fields of applied research, according to its perspective: On the one hand, within biochemistry, that of the identification of serum antibodies against lipids in multiple sclerosis (MS) as potential biomarkers for the diagnosis of this pathology; and on the other, within immunological assays, with their materials and methods, that of ELISA-type enzyme-linked immunosorbent assays for the detection of antibodies against autoimmune diseases, as improved diagnostic techniques for these diseases.
    [0016] STATE OF THE TECHNIQUE.-
    [0018] Multiple sclerosis (MS) is a demyelinating and neurodegenerative inflammatory disease of the central nervous system (CNS) that begins in most cases in the third and fourth decade of life, and therefore entails a great socioeconomic cost. These patients have a relapsing course in the relapsing remitting phase (RRMS), which cause motor and sensory alterations, but after a variable period they develop the progressive secondary form (SPMS) in which the evolution is continuous. Approximately 50% of patients take a long time to develop this form of the disease, but the rest will need a wheelchair after 15 years of evolution. A minority group of patients develop the disease in the sixth or seventh decade of life and the progression is continuous from the beginning, but this form of the disease, primary progressive (PPMS), is believed to be actually another pathology (Ref. bibliographic 1).
    [0020] The etiology of the disease is not known, although the most accepted theory is that it is an autoimmune disease in which B lymphocytes play a major role (2-5). Histological studies carried out by the research group and others have demonstrated the presence of IgG and IgM class antibodies in lesions around axons and oligodendrocytes that are associated with activated complement and macrophage activating factors (6-8).
    [0022] In this sense, various clinical trials in which anti-B lymphocyte treatments have been tested, such as anti-CD20 monoclonal antibodies (rituximab, ocrelizumab, and ofatumumab), anti-CD19 (inebilizumab), and blockers of the BAFF / APRIL pathway (atacicept , belimumab, and LY2127399) have shown high efficacy (3, 5, 9, 10), highlighting the relevance of B cells in MS.
    [0023] There is also no specific diagnostic test for the disease to date, which is diagnosed based on the characterization of clinical signs and symptoms compatible with alterations of the white matter separated in time and space together with the data obtained with complementary tests such as nuclear magnetic resonance. (NMR) and detection of intrathecal synthesis of IgG (SlIgG).
    [0025] Diagnostic criteria based on magnetic resonance imaging (11) have been reviewed over recent years (12) and it has been shown that they have a sensitivity of 80% and a specificity of 70% (13), so that apply them wisely to avoid false diagnoses (14). In order to obtain this sensitivity, intravenous injection of gadolinium is also necessary and, not least, it is an expensive technique, which contributes to the great socioeconomic cost of MS (Fernández O. et al. 2017).
    [0027] The most sensitive technique to analyze the presence of SllgG is the detection of oligoclonal IgG bands in cerebrospinal fluid (CSF) (15), based on the performance of an isoelectric focusing to separate the immunoglobulins present in paired samples of serum and CSF and subsequently a western blot, which is complex and requires highly specialized personnel for its interpretation. Therefore, it is difficult to standardize and there is a lot of variability in the sensitivity achieved between laboratories, ranging from 91% to only 69% (16). In addition, for the development of this technique, a lumbar puncture is required to obtain the CSF sample, which entails great logistics (specialist doctors, puncture room, puncture material ...) and can cause the patient to severe headaches and even neurological damage and therefore it is not performed in countries like the USA.
    [0029] Due to all these problems, numerous studies in serum have been attempted to detect antibodies against CNS proteins. For example, the patent document with publication number US2005064516-A1 refers to various molecules used as markers of multiple sclerosis, among which is the p chain of IgM, and in claim 3, it is indicated that the protein can be isolated from blood serum. Furthermore, analyzes of B lymphocytes have shown a relationship between blood B lymphocytes and in the central nervous system (17-21). In fact, antibodies isolated from the serum of these patients cause damage to oligodendrocytes and myelin (22).
    [0030] Antibodies have been detected around myelin, oligodendrocytes, and axons, but the relative contribution of B cells in the blood and in the CNS to the production of these antibodies is not yet clear (23). However, it is known that one of the hallmarks of MS is the presence of intrathecal synthesis of IgG in more than 90% of patients (24). In fact, the relevance of oligoclonal bands (OCB) has been reinforced in recent diagnostic criteria for MS (25). The high prevalence of antibodies and OCB in the CNS of patients with MS has motivated multiple studies to identify antigens directed by the antibody response in MS, which have concluded that lipids, as the main components of the myelin layer of the nerve fibers are the main target of the autoimmune response in MS (26).
    [0032] Serum antibodies have been detected against gangliosides and cerebrosides, fundamental components of axons and myelin (27,28). The data are indexed in the work of Antel J, Bar-Or A. (27), and show that antibodies against gangliosides in serum have been detected in between 50% and 13% of the MS patients analyzed and in almost one 40% of patients with other autoimmune.
    [0034] The latest microarray techniques have confirmed that there is an increase in the levels of antibodies to lipids in the serum of these patients, which are also associated with different types of the disease (29). Analysis of cerebrospinal fluid (CSF) samples using these array techniques detected antibodies to lipids in the CSF of MS patients (30, 31). In fact, IgG antibodies against sulfatides, ganglioside GM4, and galactocerebroside have been detected in multiple studies (32-35) and have been shown to worsen experimental autoimmune encephalomyelitis (EAE), an experimental model of MS (30). It should also be noted that the detection of IgM in the CSF against phosphatidylcholine (PC) is associated with a more severe disease course in MS (32, 33, 36 38), and IgG antibodies against lactosylceramide (LC) in serum they are also associated with brain tissue damage in MS patients (39).
    [0036] For these reasons, the intrathecal production of IgM and IgG antibodies against phosphatidylcholine (PC) and lactosylceramide (LC) has recently been associated with disease activity and neurodegeneration, suggesting a potential role for these antibodies against corresponding lipids as biomarkers in MS.
    [0038] In view of this, the work that has given rise to the present invention has had as its first objective the detection and quantification of IgG and IgM antibodies against LC and PC in serum samples from patients with MS, for which it has been necessary to resort to to an ELISA technique improved, high sensitivity, which solves the problems associated with lipid management and the low affinity and concentration of said antibodies in the blood, revealed for example in studies such as that of Jurewicz et al. (40), in which an increase in IgM class antibodies against cholesterol has been seen by ELISA, but its role as a diagnostic biomarker has not been demonstrated, as indicated in the section on how to perform it in the comparison of results with those of the improved technique ( Figures 1 and 2 ).
    [0040] Another notable antecedent is that of the work of Carlos J. Bidot et al. (41), in which the ELISA technique is applied in the detection of IgM and IgG antibodies in patients in different stages of MS using a panel of several phospholipid antigens, including phosphatidylcholine (PC), although IgM-PC does not appear as A single phospholipid marker, but rather as a marker to be selected from a set of six targets, and as with the previous case, the technique does not allow obtaining results demonstrating a diagnostic biomarker capacity, neither for this nor for the other possible markers; Rather, the objective of this work is to assess neuroimaging characteristics in MS patients with antiphospholipid antibodies in plasma.
    [0042] On the other hand, with the new adapted ELISA, it has been found, according to the experimental results that are set out below within the compendium of the invention, that serum IgM antibodies against PC (IgM-PC) are surprisingly high in patients with MS, and that there is also an association with the stage of the disease: patients with CIS and RRMS exhibit higher levels of IgM-PC than SPMS, PPMS and BENMS, without, on the contrary, significant differences in serum IgG antibodies having been detected against PC (IgG-PC), nor IgM or IgG against LC (IgM-LC and IgG-LC, respectively) between MS patients and controls, which reveals serum IgM-PC antibodies as diagnostic biomarkers of multiple sclerosis and the different phases of this pathology.
    [0044] Comparing the ultrasensitive ELISA technique developed for the indicated objective with previous studies, it is worth highlighting the following differences that have made it possible to have successfully selected serum IgM-PC as a diagnostic biomarker for MS:
    [0046] 1 ° Specificity in its application: Unlike previous ELISA applications aimed at the analysis of different antigens (gangliosides, cerebrosides, lactosylceramide, cholesterol), with the implemented ELISA, the presence of IgM antibodies against phosphatidylcholine in serum has been analyzed for the first time .
    [0047] 2 ° High sensitivity: In the aforementioned work by Jurewicz et al., For example, the antibodies have to be isolated, and even so it can be observed that the readings obtained do not reach 0.15 OD, when with the new procedure there are readings of up to 0.8, which are at the high limit of the ELISA technique, indicating an ability to detect very low amounts of antibodies to lipids.
    [0049] 3 ° High precision in the diagnosis of MS: The sensitivity obtained in the studies carried out previously does not reach 50%, while now, after analyzing more than 350 patient sera, it has been found that approximately 90% of the patients In the first outbreak or with the secondary progressive form of the disease, they present IgM antibodies against PC in serum, which is the most precise biomarker described to date for the diagnosis of MS.
    [0051] This has been possible because the main impediments that until now have made the standardization and interpretation of the existing similar ELISAS difficult have been overcome, such as: the faulty adhesion of the lipid to the plate, which has been solved by adjusting concentration and solvent; blocking, avoided by using a solution that does not bind to lipids, unlike those used classically; the risk of solobilization of lipids, minimized by cold washing; or the low affinity and concentration of antibodies in the blood, corrected by amplification of the signal using a biotinastreptavidin system and TMBone as substrate, all this to give rise to the new semi-quantitative technique in thirteen steps, specific for the proposed marker, which is explained in the next section in detail.
    [0053] Various patent documents are known about ELISA-type immunoassays that, as is foreseeable, apply some of the steps of the proposed method for the detection of other molecules, in some cases similar (IgG, Igs in general), although there is no record of any specifically applied to IgM-PC. Notable among these patents are the North American US2007020691-A1, which describes a diagnostic method for MS or a demyelinating disease, which uses, among other possibilities, an ELISA technique for lipids, coating immunoassay plates with gangliosides and cardiolipin, blocking with diluent of assaying and detecting antibody (IgG) conjugated to HRP; and Japanese patent JPH06148193A, referring to a vehicle to bind an antiphospholipid antibody, used for the diagnosis of autoimmune diseases, as well as an immunoassay method that uses said vehicle, a method that presents some parts similar to the one developed, although not for IgM, such as the use of methanol and evaporation to bind the antibody to the plate.
    [0054] However, there is no known immunochemical procedure that combines all the steps of the proposed sequence of the invention, nor that applies all the resources and specific conditions in which it is carried out, which influence the high selectivity and precision achieved, and which enable its standardization.
    [0056] COMPENDIUM OF THE INVENTION.-
    [0058] In view of the prior art, the determination of the levels of IgM against phosphatidylcholine (IgM-PC) and against lactosylceramide (IgM-LC) by means of the new semi-quantitative ELISA implemented in serum samples from patients with multiple sclerosis (MS ) and in controls in cohorts from the USA and Spain, with the results reflected in the graphs of Figures 1 and 3 , make the serum IgM-PC antibody a biomarker for MS, and as such it is claimed to be an invention, since an increase in the level of IgM-PC antibodies in the serum of patients with this disease of up to 0.8 OD (mean Optical Density) is achieved, with reactivity indices P = 0.004 and P = 0.0004 with respect to controls, which triples the value of each cohort group (Figure 1), and doubling the level of IgM-LC antibodies in the serum of the same patients (Figure 2), which represents an impressive increase in serum IgM-PC antibodies detected in MS patients.
    [0060] Likewise, the detection of different serum levels of IgM-PC in patients with isolated clinical syndrome (CIS), relapsing remitting multiple sclerosis (RRMS), secondary progressive (SPMS), primary progressive (PPMS) and benign (BEN), in comparison with the controls, according to the results reflected in Figure 3, they confer on the serum IgM-PC antibody the ability to associate with the course and subtype of MS, and make it a biomarker of the different phases of the disease, which also means a remarkable novelty. Specifically, after the semi-quantitative ELISA test carried out, increases in the level of IgM-PC antibodies were obtained in sera from patients with CIS, RRMS, SPMS and primary PPMS, around 0.3, 0.25, 0.2 and 0.15 respectively, compared to an increase in this antibody around 0.05 for the cohort group (graph in Figure 4 ), translated into a percentage of positive sera of 88.2%, 88.7%, 58.0% and 59.5% respectively, compared to 25.0% for the cohort group .
    [0062] The higher levels of IgM-PC antibodies in the serum of patients during the CIS and RRMS phase of the disease mean that IgM-PC serum is an ideal biomarker for the early diagnosis of MS.
    [0063] All of which justifies the use of serum IgM-PC as a biomarker for MS, alone or in combination with other agents, in the preparation of commercial compositions and detection systems for the differential diagnosis by immunochemical techniques of this disease and its different stages, of clinical isolated syndrome (CIS), relapsing remitting multiple sclerosis (RRMS), secondary progressive (SPMS), primary progressive (PPMS) and benign (BEN), particularly important for the early phases of CIS and RRMS, and not only due to the ultrasensitive ELISA method adapted for the specific detection of said antibody, but by any other immunochemical technique that may be successfully developed for this purpose in the future.
    [0065] In this sense, the semi-quantitative ELISA technique developed for the specific detection of the IgM-PC antibody in serum samples from potential MS patients is ideal as a method for diagnosing the disease, due to its commented high sensitivity and precision compared to existing similar ELISAS. so it is also a claim of invention.
    [0067] This has been possible because the problems inherent to this type of analytics have been solved, as follows:
    [0069] Low solubility of lipids in aqueous solvents and difficulty of sticking to the ELISA plate:
    [0071] • Methanol is used as a diluent for the lipids tested.
    [0073] Low binding of lipids to the ELISA microtiter plate:
    [0075] • Greiner high-binding plates are used that have a high capacity to bind different molecules.
    [0077] • The plates are incubated overnight at 4 ° until complete evaporation of the methanol to allow the lipid to adhere to the microtiter plate).
    [0079] • To prevent the lipid from detaching from the plate, PBS (physiological saline buffered with phosphate pH 7.2 at 4 ° C is used as a wash solution.
    [0081] Classic blocking solutions bind to lipids and thus prevent the binding of specific antibodies to these antigens present in the analyzed samples:
    [0082] • Albumin and other similar proteins are used to block the sites that remain free on the microtiter plate after lipid bonding and thus prevent non-specific bonding of serum antibodies to the plate, and thus erroneous readings.
    [0084] • These proteins that are classically used in the ELISA technique are lipid transporters, and therefore they bind to lipids that have previously been attached to the microtiter plate, preventing antibodies against lipids present in serum from being able to bind to them.
    [0086] • To avoid this blockage of antigen-antibody binding, a blocking solution that does not bind to lipids is used: SuperBIock ™ (PBS) Blocking Buffer (Thermo Fisher Scientific).
    [0088] Low affinity of antibodies against lipid antigens and low serum concentration of the same:
    [0090] • A signal amplification system is used based on the use of a secondary antibody anti Human IgM, anti human IgM biotinylated (Jackson ImmunoResearch), followed by streptavidin-peroxidase, and on the use as substrate of 1-Step ™ Ultra TMB -ELISA Substrate Solution (Thermo Scientific ™), which is the most sensitive to date. Biotin binds about 12 streptavidins and therefore each serum IgM antibody that binds to the lipid will bind 12 peroxidases, which oxidize its substrate and thus the amplified signal is obtained.
    [0092] The new ultra-sensitive ELISA technique designed for the specific detection of serum IgM-PC antibodies consists of the following steps that include the indicated procedural parameters and resources:
    [0094] 1) Add phosphatidylcholine (Merck) 1 pg / well diluted in 100 microliters of methanol as diluent in Greiner High-Binding microtiter plates (Greiner), of high molecular binding capacity:
    [0096] 2) Incubate the plates overnight at 4 ° C until complete evaporation of the methanol to ensure adherence of the lipid to the plate;
    [0097] 3) Wash 5 times with 250 µl / well of physiological serum buffered with phosphate pH = 7.2 at 4 ° C, thereby preventing the lipid from detaching from the plate;
    [0099] 4) Add 200 µl of the SuperBlock ™ (PBS) Blocking Buffer ThermoFisher) (BF) solution, as a specific blocking solution, and incubate for 2 hours.
    [0101] 5) Repeat stage 3.
    [0103] 6) Add the samples at 1/100 dilution in BF and incubate for 2 hours at room temperature.
    [0105] 7) Repeat stage 3.
    [0107] 8) Add 100 µl of biotinylated anti human IgM (Jackson ImmunoResearch) diluted 1 / 20,000 in BF, as secondary antibody for signal amplification, and incubate for 2 hours at room temperature.
    [0109] 9) Repeat stage 3.
    [0111] 10) Add 100 µl of streptavidin-peroxidase (Jackson ImmunoResearch) diluted 1/1000 in BF and incubate for 30 minutes at room temperature.
    [0113] 11) Repeat stage 3.
    [0115] 12) Add 100 µl of 1-Step ™ Ultra TMB-ELISA Substrate Solution (Thermo Scientific ™) and incubate for 5 minutes.
    [0117] 13) Add 100 µl of H2SO4 diluted 5% in distilled water to cut the reaction and read the plate in a spectrophotometer at 450 nm.
    [0119] This ultrasensitive technique has demonstrated for the first time the presence of a biomarker in the blood for the early diagnosis of MS, which is of great clinical relevance since the sooner treatment of the disease begins, the better its evolution. It also has the advantage over existing diagnostic methods that no CSF fluid is analyzed, reducing protocols and patient time in hospital, who does not suffer side effects derived from the procedure.
    [0120] Methodologically it also has advantages over the existing ones. It is quantitative and therefore objective, and although the protocol has been developed manually, it can be automated, which would allow maximizing resources and reducing costs, as well as making it standardizable and reproducible in any laboratory.
    [0122] In short, it is a technique that can be implemented in hospital or extra-hospital clinical diagnostic laboratories worldwide.
    [0124] FIGURES AND GRAPHICS.-
    [0126] At the end of this descriptive report, the following figures are presented with graphs that reflect the results of the tests carried out with the new semi-quantitative ELISA in patients with MS for the determination of the levels of IgM-PC and IgM-LC, including for comparative purposes , the graph resulting from the analysis by Jureviwicz et al. for IgM against cholesterol:
    [0128] Figure 1: Graphs of levels of IgM versus phosphatidylcholine (IgM-PC) in serum samples of patients from USA and Spain with MS and controls.
    [0130] Figure 2: Graph of IgM levels versus cholesterol in the test carried out by Jureviwicz et al. by conventional ELISA.
    [0132] Figure 3: Graphs of IgM levels versus lactosylceramide (IgM-LC) in serum samples from patients with MS in the USA and Spain and controls.
    [0134] Figure 4: Graph of serum levels of IgM-PC in patients with different phases of MS: CIS, RR, SP, PP and BEN, and control group C.
    [0136] Figure 5: Graph of serum levels of IgM-LC in patients with different phases of MS: CIS, RR, SP, PP and BEN, and control group C.
    [0138] The graphs of these figures are discussed in the next section, describing how to carry out the invention.
    [0140] FORM OF REALIZATION.
    [0141] The presence of serum IgG and IgM antibodies against lactosylceramide (LC) and phosphatidylcholine (PC) has been analyzed with the new ultrasensitive semi-quantitative technique developed.
    [0143] 1. Materials and Methods.-
    [0145] 1.1. Serum samples.-
    [0147] Peripheral blood samples were collected from 362 MS patients and 80 patients without neurological diseases (control group). Serum samples were aliquoted and stored at -80 ° C until analyzed.
    [0149] The samples were obtained from the Longitudinal Comprehensive Research on Multiple Sclerosis (CLIMB, Boston, MA) and EPIC (San Francisco, CA), as part of the SUMMIT consortium and the Neurology Service of the Virgen del Rocío Hospital (Seville, Spain). All participants gave their written informed consent for their participation in the study. The patients were relapse-free at the time of blood sampling. and were classified as clinically isolated syndrome (CIS, n = 17), relapsing remitting MS (RRMS, n = 62), secondary progressive MS (SPMS, n = 50), primary progressive MS (PPMS, n = 37) and benign MS (BENMS, n = 36).
    [0151] The clinical and demographic data of the MS patients and the control group are summarized in Table 1.
    [0153] Table 1.
    [0156] In this table the fields "Women" indicate the number of cases analyzed and percentage in the group, "Age" the duration of the disease and the expanded disability status scale (EDSS): mean, minimum and maximum; and "Treatment" the number and percentage of patients treated.
    [0158] The samples from the patients whose test results are reflected in Figure 1 were treated with IFN, glatiramer acetate, and natalizumab. The treatment used for samples from patients with relapsing remitting form (RR) was interferon beta (IFN); those of patients with the secondary progressive form (SP) were treated with IFN, glatiramer acetate, and natalizumab, and patients with the benign form were treated with IFN and natalizumab. Primary progressive (PP) were not treated.
    [0160] 1.2. ELISA.- Assay
    [0162] To detect IgG and IgM antibodies against lactosylceramide (LC) and phosphatidylcholine (PC), the 96-well plates were incubated with the corresponding antigen. Plates were washed three times with phosphate buffered saline (PBS) and incubated with blocking solution. Serum samples were diluted in blocking solution and pipetted into the wells.
    [0164] Antibodies bound to antigens were detected with corresponding secondary antibodies (Jackson Immunoresearch), followed by avidin-peroxidase (Sigma-Aldrich). Finally, TMBone (Kementec) was used as a substrate. Plates were read at 450 nm using an Infinite® 200 PRO spectrophotometer (Tecan instrument). Serum samples were classified as positive when the O.D was greater than the mean ± 2xSEM (standard error of the mean) of the control group.
    [0166] 1.3. Statistics.-
    [0168] Statistical analyzes were performed with the GraphPad Prism statistical package (version 6.0). P values less than 0.05 were considered statistically significant. The Mann-Whitney U test was used for comparisons between two groups and the Kruskal-Wallis test for comparisons between more than two groups. To analyze the percentage of individuals who showed antibodies against lipids, Fisher's test was used for comparisons between patients with MS and the control group, and Pearson's Chi-square test for comparisons between different types of EM.
    [0169] 2. Results.-
    [0171] Two relevant results:
    [0173] First.- Increase in serum of IgM antibodies against phosphatidylcholine in patients with MS:
    [0175] Abnormalities in sphingolipid metabolism have been described in MS (42-44). In addition, anti-lipid antibodies are detected in patients with SPMS and correlate with markers of disease progression, such as brain atrophy (30, 39, 45). Therefore, serum IgG and IgM antibodies against PC and LC were initially analyzed in MS patients and controls in independent cohorts from the United States of America (USA) and Spain. A higher reactivity of IgM against phosphatidylcholine (IgM-PC) was detected in serum samples from patients with MS as reflected in Figure 1 . On the contrary, no significant differences were detected in the serum levels of IgM against lactosylceramide (IgM-LC) nor in the levels of IgG against PC (IgG-PC) or LC (IgG-LC) among patients with MS. and controls, as shown in Figure 3 .
    [0177] The graphs in Figure 1 reflect the levels of IgM versus phosphatidylcholine (IgM-PC) in serum samples from patients with MS and controls, obtained using the ultrasensitive ELISA technique developed. The data are presented as the mean optical density (OD) ± SEM (standard error of the mean), and it can be observed that patients with MS have much higher levels of IgM antibodies against phosphatidylcholine than controls in both sections, obtaining values of up to 0.8 OD
    [0179] The efficacy of the new technique is shown when the results are compared with those of the graph in Figure 2 , of the analysis of the presence of serum IgM antibodies against cholesterol in a patient with MS carried out by Jureviwicz et al. using conventional ELISA, in which, although it is observed that MS patients have more antibodies against said antigen, values are obtained in all cases that do not exceed the level of 0.2 OD, which is insufficient for an IgM-Cholesterol function as a biomarker of MS.
    [0181] And also compared to the graphs in Figure 3 , which reflect the levels of IgM against lactosylceramide (IgM-LC) in serum samples from patients with MS and the control group (C) (NS: not significant), where the values OD are half that of IgM-PC.
    [0182] Second.- IgM-PC antibodies are associated with the course and subtype of the disease:
    [0184] The progression of MS is believed to involve a change in the inflammatory process that would shift from being mediated by peripheral immune cells to being caused by cells resident in the CNS (46, 47). Therefore, the presence in serum of IgM against phosphatidylcholine (IgM-PC) was analyzed in patients with different phases of the disease, detecting higher serum levels of IgM-PC in patients with isolated clinical syndrome (CIS), multiple sclerosis Recurrent sender (RR), secondary progressive (SP), primary progressive (PP) compared to controls (C), as reflected in Figure 4 , in which the data are presented as the mean optical density (OD ) ± SEM (standard error of the mean). NS: not significant. In addition, patients classified as CIS and RRMS show higher serum levels of IgM-PC antibodies than SPMS and PPMS, as is also confirmed by the percentages of positive sera for IgM-PC in patients with MS and the control group shown in the Table 2. No significant differences were found in serum levels of PC-IgM between the samples of patients with CIS and RRMS, nor between those of patients with SPMS and PPMS.
    [0186] Additionally, no differences were detected in serum levels of LC-IgM when samples of different MS subtypes were analyzed, as reflected in the graphs in Figure 5 and the percentage of positive sera for LC-IgM in patients with MS and control group shown in Table 2. Nor were differences detected in serum levels of IgG-LC or IgG-PC.
    [0188] Table 2 shows the percentages of positive sera for IgM-PC and IgM-LC in patients with different subtypes of MS and control group.
    [0190] Table 2.
    [0193] In this table, IgM-PC: IgM-PC positive sera; IgM-LC: Positive sera for IgM-LC; IgM-LIP: Positive sera for IgM-PC or IgM-LC; CIS: clinically isolated syndrome; RR: remission-remission; SP: progressive secondary; PP: progressive primary; BEN: benign; PC: phosphatidylcholine; LC: lactosylceramide; LIP: lipids.
    [0195] 3. Discussion of the results.-
    [0197] Intrathecal synthesis of IgG is detected in the majority of MS patients (24) and more than 60% of these patients have IgM with lipid-reactive CSF (32). In fact, the detection of lipid-reactive IgM OCM in CSF is considered a poor prognostic marker in MS (36-38). IgG and IgM antibodies to myelin proteins have also been detected in the serum of MS patients (48, 49). However, the detection of lipid-reactive antibodies is complicated due to technical problems related to the solubility of the antigen, the accessibility of reactive groups, and the requirement for auxiliary lipids (50, 51). The work carried out describes a new technique for the analysis in patients with MS of serum antibodies against PC and LC. Using this assay, increased levels of IgM-PC have been detected in serum samples from patients with MS, with no significant differences between the control group and patients with MS when the presence of IgM-LC was analyzed, which corroborates the Previous studies, developed using protein arrays, did not observe an increase in serum IgM antibodies against LC in patients with MS (39).
    [0199] The presence in blood of B cells specific to central nervous system antigens has been associated with the occurrence of relapses (52). In addition, B-cell targeted therapies slow disease progression (53). In the research that led to the present patent application, higher serum levels of IgM-PC have been detected in patients with CIS and RRMS than SPMS and PPMS, which suggests that serum IgM-PC reflects the development of the inflammatory process and destruction of myelin caused by peripheral blood B lymphocyte activation. In addition, these data are in line with previous studies, in which a decrease in the humoral immune response and a predominant role for the intrinsic mechanisms of the CNS during the progressive phase of MS have been observed (46, 54). In fact, we previously described that OCBs of IgM against lipids in CSF persist during the course of the disease (36), and we also detected IgM and IgG antibodies deposited in axons and oligodendrocytes in MS CNS samples (55), suggesting that these antibodies are produced locally in the CNS, probably in detectable follicle-like structures in the meninges (56 59). Although some lipid-reactive antibodies have been shown to harbor pathogenic activities (30), it is not yet clear whether IgM-PC antibodies harbor direct pathogenic activity on myelin, which could also cause the release of bioactive lipids (8, 45, 60, 61), and / or are simply by-products of the inflammatory process that have a role as a diagnostic biomarker (31, 34, 39, 61).
    [0201] In summary, all these data indicate that the detection of serum IgM-PC is at least one diagnostic marker of the disease and can also complement another prognostic marker, such as the detection of antibodies in samples of cerebrospinal fluid (38).
    [0203] IgM against PC is part of the natural antibody repertoire, which provides protection against multiple pathogens (62-64). Natural autoantibodies are produced by B-1 cells, which are T-independent (65, 66) and generally do not undergo an isotype switch to IgG (67). The mechanism involved in the generation of natural antibodies is the binding of the specific antigen to the B-cell receptor (BCR), and also through additional B-cell stimulation pathways (68). It has been shown that PC can activate various B-cell pathways (69, 70), and it is possible that myelin damage releases PC into the blood and, in an inflammatory setting, activates peripheral B-1 lymphocytes specific for this antigen through the BCR and other channels. In fact, increases in serum of lipids such as GM1 and GD1a, fundamental components of myelin, have been observed in patients with RRMS during the first flare (71). In addition, the finding resulting from the investigation carried out on the increase in serum of IgM-PC suggests an exacerbated activity of B-1 cells, which could be a fundamental mechanism in the pathogenesis of MS (23), as described in other autoimmune diseases such as rheumatoid arthritis or idiopathic thrombocytopenic purpura (72). In fact, the number of B-1 cells in cerebrospinal fluid has been associated with a poor prognosis in MS patients (38).
    [0205] In conclusion, the results obtained identify IgM-PC antibodies as diagnostic biomarkers of MS, and suggest a role for B1 cells in the pathogenesis of the disease. Future studies should extend these observations to additional cohorts of patients, analyze the effect of disease-modifying therapies on IgM-PC antibodies, and the value of B1 cells as therapeutic targets in MS.
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    权利要求:
    Claims (13)
    [1]
    1. Serum IgM antibody against phosphatidylcholine (IgM-PC) as a biomarker for multiple sclerosis (MS), selected from the group of serum antibodies against lipids detected in the autoimmune response of MS, particular case of IgM against lactosylceramide ( IgM-LC), characterized by achieving an increase in the level of IgM-PC antibodies in the serum of patients with MS of up to 0.8 OD (Average Optical Density) by semi-quantitative ELISA test, with reactivity indices P = 0.004 and P = 0.0004 with respect to controls in cohorts from the United States and Spain, which triples the value of the cohort group in both cases (graph in Figure 1) , and that doubles the level of IgM-LC antibodies in the serum of the same patients with said disease (graph of Figure 2).
    2. Serum IgM-PC antibody as a biomarker for MS, according to claim 1, characterized by its ability to associate with the course and subtype of the disease, by obtaining, after semi-quantitative ELISA test, increases in the level of IgM-PC antibodies in sera from patients with isolated clinical syndrome (CIS), relapsing remitting multiple sclerosis (RRMS), secondary progressive (SPMS) and primary progressive (PPMS), around 0.3, 0.25, 0.2 and 0.15 respectively, compared to an increase in this antibody around to 0.05 for the cohort group (graph of Figure 3), translated into a percentage of positive sera of 88.2%, 88.7%, 58.0% and 59.5% respectively, compared to 25.0% for the cohort group.
    3. Use of serum IgM-PC as a biomarker for MS, according to claim 1, alone or in combination with other agents, in the preparation of compositions and commercial detection systems for differential diagnosis by immunochemical techniques of said disease.
    4. Use of serum IgM-PC as a biomarker for the CIS, RR, SP and PP phases of MS, according to claims 1 and 2, alone or in combination with other agents, in the preparation of commercial compositions and detection systems for differential diagnosis by immunochemical techniques of these phases of the disease.
    5. Use of serum IgM-PC as a biomarker for the CIS and RR phases of MS, according to claims 1 and 2, alone or in combination with other agents, in the preparation of commercial compositions and detection systems for differential diagnosis using immunochemical techniques of these early stages of the disease.
    6. High sensitivity semi-quantitative ELISA technique for the detection of serum IgM antibodies against phosphatidylcholine (IgM-PC) in patients with multiple sclerosis (MS), of the type of ELISA used for the detection of antibodies against antigenic substances and compounds in samples serum from patients with autoimmune diseases, characterized by a procedure consisting of the following stages or steps:
    1) Add phosphatidylcholine (Merck) 1 pg / well diluted in 100 microliters of methanol as diluent in Greiner High-Binding microtiter plates (Greiner), of high molecular binding capacity:
    [2]
    2) Incubate the plates overnight at 4 ° C until complete evaporation of the methanol to ensure adherence of the lipid to the plate;
    [3]
    3) Wash 5 times with 250 µl / well of physiological serum buffered with phosphate pH = 7.2 at 4 ° C, thereby preventing the lipid from detaching from the plate;
    [4]
    4) Add 200 µl of the SuperBlock ™ (PBS) Blocking Buffer ThermoFisher) (BF) solution, as a specific blocking solution, and incubate for 2 hours.
    [5]
    5) Repeat stage 3.
    [6]
    6) Add the samples at 1/100 dilution in BF and incubate for 2 hours at room temperature.
    [7]
    7) Repeat stage 3.
    [8]
    8) Add 100 µl of biotinylated anti human IgM (Jackson ImmunoResearch) diluted 1 / 20,000 in BF, as secondary antibody for signal amplification, and incubate for 2 hours at room temperature.
    [9]
    9) Repeat stage 3.
    [10]
    10) Add 100 µl of streptavidin-peroxidase (Jackson ImmunoResearch) diluted 1 / 1,000 in BF and incubate for 30 minutes at room temperature
    [11]
    11) Repeat stage 3.
    [12]
    12) Add 100 µl of 1-Step ™ Ultra TMB-ELISA Substrate Solution (Thermo Scientific ™) and incubate for 5 minutes.
    [13]
    13) Add 100 µl of H2SO4 diluted 5% in distilled water to cut the reaction and read the plate in a spectrophotometer at 450 nm.
    7. High sensitivity semi-quantitative ELISA technique for the detection of serum IgM-PC antibodies in patients with MS, according to claims 1 to 6, applied as a method of differential diagnosis of MS and of the CIS, RR, SP and PP phases of this disease.
    8. High sensitivity semi-quantitative ELISA technique for the detection of serum IgM-PC antibodies in patients with MS, according to claims 1 to 6, applied as a method of differential diagnosis of MS and the early CIS and RR phases of this disease.
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    WO2010121714A1|2009-04-24|2010-10-28|Diametra S.R.L.|Complexes between phospholipids and protein vimentin, and in vitro methods for the detection of antibodies against these complexes|
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