ITFI20110070A1 - METHOD AND DIAGNOSTIC KIT FOR MULTIPLE SCLEROSIS - Google Patents

Description

PATENT APPLICATION FOR INDUSTRIAL INVENTION WITH THE TITLE:

METHOD AND DIAGNOSTIC KIT FOR MULTIPLE SCLEROSIS

FIELD OF INVENTION

The present invention relates to the field of diagnosis and / or prognosis methods; in particular it refers to the field of methods for the diagnosis and / or prognosis of multiple sclerosis and / or monitoring the efficacy of therapy.

STATE OF THE ART

Multiple sclerosis (MS) is one of the most common diseases affecting the central nervous system (CNS). It is a chronic, progressive and disabling disease characterized by the destruction of the myelin sheath surrounding the nerve fibers, a process called “demelination”. It is a multifactorial pathology, whose onset is given by the combination of a genetic susceptibility with environmental factors; an autoimmune origin is hypothesized, in genetically predisposed subjects, triggered by the encounter with an exogenous antigen.

Multiple sclerosis affects about 2 million people in the world, of which almost half a million in Europe and more than 50,000 in Italy; among neurological disorders it is the most common among young adults and the main neurological cause of disability. About 1,800 new cases occur in Italy every year. The incidence is about 3-4 in 1000 affected individuals. The disease typically occurs between the ages of 14 and 40, with a peak around the age of 30; On the other hand, it is infrequent beyond the age of 50.

The great variability of symptoms that characterize multiple sclerosis is a consequence of the degeneration process of the myelin that lines the nerves, which causes the blocking or slowing of communication between the various parts of the individual's nervous system. The disease can affect any area of the central nervous system, therefore from a clinical point of view it is characterized by a wide variety of neurological signs and symptoms. These can manifest themselves singly or in concomitance with the others and their appearance is gradual in terms of number and severity. In most patients with multiple sclerosis, the signs and symptoms tend to appear and disappear over time, especially in the initial phase of the disease, that is, in the first years.

The diagnosis of multiple sclerosis is formulated by the neurologist, based on a set of data obtained from various clinical investigations, accompanied by instrumental and laboratory tests. In fact, at the moment an examination is not available that alone is able to confirm the diagnosis of the disease in a certain and indisputable way; only the set of data collected and precise monitoring allow to confirm or exclude its presence and its possible progression. Therefore, the development of a diagnostic method and a related specific kit for the diagnosis of multiple sclerosis is particularly important.

It is extremely important to be able to develop new diagnostic methods for multiple sclerosis, in order to speed up diagnosis and obtain increasingly reliable prognostic indications; in fact, an early therapeutic intervention is essential in order to limit neurological damage in sick subjects. With this in mind, clinical research is moving towards the identification of specific biological markers of the disease, to be used for diagnostic and prognostic purposes. The peculiarities of a biomarker should be reproducibility, high sensitivity and specificity for the pathology; in fact, it is very difficult to find a marker that meets these requirements, especially in the case of a multifactorial disease such as multiple sclerosis. Furthermore, due to the complexity of this pathology and the heterogeneity of its manifestations, a single biomarker may not be sufficient to develop a diagnostic test, therefore the efforts of researchers are moving towards the definition of a panel of markers.

The biological molecules that are best suited to be used as biomarkers are proteins; in fact they are the molecular effectors of cellular functions, whose qualitative and / or quantitative variations reflect the physiological or pathological state of an organism. Therefore proteomics analysis seems to be the most suitable tool for the identification of such markers. In particular, the identification of biomarkers in multiple sclerosis, given its etiopathogenetic characteristics, is mainly based on the study of molecules of the immune system, which reflect immunological activation, or factors associated with neurodegeneration, which correlate with clinical aspects .

The diagnostic potential of a biomarker is associated with the possibility of being able to detect it in the samples under examination, preferably non-invasive or minimally invasive and for this reason biological fluids such as urine, blood and cerebrospinal fluid represent the best source of candidate molecules as biomarkers .

The purpose of the present invention is to provide a method for the diagnosis and / or prognosis of multiple sclerosis which is based on the identification, in a biological fluid such as blood or cerebrospinal fluid, of a specific and selective biomarker. invention is to provide a diagnostic kit for the identification of said biomarker in a biological fluid such as blood or cerebrospinal fluid.

DEFINITIONS AND ABBREVIATIONS ELISA: Enzyme-Linked ImmunoSorbent Assay

SUMMARY OF THE INVENTION

It was surprisingly found that multiple sclerosis patients have autoreactive anti-transferrin antibodies in cerebrospinal fluid and serum in concentrations significantly higher than the concentrations detectable in a subject without multiple sclerosis.

The object of the present invention is therefore a method for the diagnosis and / or prognosis of multiple sclerosis, said method comprises the identification and quantification of anti-transferrin antibodies in a biological fluid.

The identification and quantification of anti-transferrin antibodies in a biological fluid may also be useful for monitoring the efficacy of a therapy for the treatment of multiple sclerosis.

A further object of the invention is a diagnostic kit comprising at least one microtiter plate on which the transferrin antigen has been immobilized. BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Proteomic maps, subjected to silver coloring, of the cerebrospinal fluid (A) and the corresponding serum (B) of a subject with multiple sclerosis. The spots corresponding to different isoforms of the same protein are linked by a solid line; arrows indicate proteins with a single shape, while the oval indicates proteins derived from the central nervous system.

Figure 2. Proteomic maps, subjected to silver staining, of the cerebrospinal fluid of a subject with multiple sclerosis (A) and of pool I after the removal of albumin and class G immunoglobulins (IgG) (B). The spots corresponding to different isoforms of the same protein are linked by a solid line; arrows indicate proteins with a single shape, while the oval indicates proteins derived from the central nervous system. The rectangles circumscribe the areas where the spots of the albumin isoforms and the light and heavy chains of IgG are found. The squares indicate the spots corresponding to the less abundant proteins in the CSF, which are absent in the first map.

Figure 3. Detail of the two-dimensional western blots (A, B, C) and of the relative portions of the proteomic map subjected to silver stain (D, E, F) of pool I (A and D), of pool II (B and E) and of the cerebrospinal fluid pool III (C and F) of subjects with multiple sclerosis. The letters on the spots indicate the self-reactive isoforms of transferrin.

Figure 4. In A detail of the two-dimensional western blot of pool II hybridized with the CSF of a subject with migraine and in B the relative portion of the proteomic map. The rectangle in B delimits the area where the transferrin isoforms are found. The sign in A indicates the position of a protein spot used as a reference for matching.

Figure 5. A) Proteomic profile, subjected to silver staining, of the preparation of transferrin purified from human plasma. B) Two-dimensional Western blot performed by hybridizing the purified transferrin map with the specific anti-transferrin antibody. C) Detail of the two-dimensional western blot performed by hybridizing the purified transferrin map with the non-depleted CSF pool III. The boxes highlight the canonical isoforms of transferrin, while the numbers and arrows highlight the non-canonical isoforms.

Figure 6. Detail of the two-dimensional western blots carried out by hybridizing the proteomic map of purified transferrin with the non-depleted cerebrospinal fluid III pool (A) and after a competition assay conducted on the same sample for 3 hours with 50Î1⁄4g / ml of transferrin (B ) or for 5 hours with 200Î1⁄4g / ml of transferrin (C). The rectangle highlights the area where the transferrin isoforms recognized by the antibody are found, while the oval indicates the most reactive spot, of which a trace remains even after the inhibition of the immunological reaction.

Figure 7. Detail of two-dimensional western blots performed by hybridizing the proteomic map of the cerebrospinal fluid IV pool after albumin and IgG depletion with the corresponding sera pool (A) or with a single serum sample from a subject with multiple sclerosis (B ). The boxes highlight the immunoreactive spots. The circular sign at the bottom indicates the position of a protein spot used as a reference for matching.

Figure 8. Detail of the two-dimensional western blots carried out by hybridizing the proteomic map of purified transferrin with two serum samples from subjects with multiple sclerosis (A and B). The rectangles indicate the immunoreactive spots that correspond to the canonical isoforms of transferrin, while the oval circumscribes those corresponding to the recognized non-canonical isoforms.

Figure 9. One-dimensional Western blots carried out by hybridizing the recombinant transferrin with the specific anti-transferrin antibody (A), with the serum of a subject with multiple sclerosis (B), with the serum of a healthy individual (C). The arrow indicates the position of the transferrin.

Figure 10. Amount of anti-transferrin autoantibody detected by ELISA assay in the serum of an individual with multiple sclerosis (serum MS) and in that of a healthy subject (serum N). The quantity of transferrin as antigen adsorbed in the wells is reported on the abscissa; the ordinate shows the absorbance values, which are proportional to the amount of autoantibody present.

Figure 11. Amount of anti-transferrin autoantibody detected by ELISA assay in the serum of 57 healthy individuals, 50 with multiple sclerosis, 21 with other neurological diseases and 21 with other autoimmune diseases.

DETAILED DESCRIPTION OF THE INVENTION

Preferably, according to the invention, the biological fluid to be analyzed is serum or cerebrospinal fluid. The serum is definitely preferable due to the less invasiveness of its withdrawal.

The assay for the identification and quantification of anti-transferrin antibodies according to the invention is preferably carried out by means of the ELISA method. Said method preferably uses transferrin antigen whose purity is high, preferably higher than 97% by weight. Transferrin purified from plasma of different species can be used, preferably human or more advantageously, recombinant transferrin whose purity is higher than 98% by weight can be used.

The kit according to the invention therefore comprises at least one microtiter plate on which the transferrin antigen whose purity is higher than 97% by weight has been immobilized. Recombinant transferrin is preferably used because it is purer. The concentration of the immobilized antigen is preferably 1.0-20 Î1⁄4g / ml (corresponding to 0.15-3.00 Î1⁄4g / cm <2>), more preferably 2.0-5.0 Î1⁄4g / ml; even more preferably 3.0-4.0 Î1⁄4g / ml.

The kit according to the invention preferably further comprises at least one container with a positive control or a sample of antitransferrin antibody.

The kit according to the invention preferably further comprises at least one container with a negative control or for example a sample of bovine serum albumin.

The kit according to the invention preferably also comprises at least one container containing a buffer solution to be used for the washing steps of the wells and for the preparation of the solutions with which to dilute the samples to be analyzed and the secondary antibody, preferably PBS-T (137mM sodium chloride, 2.7mM potassium chloride, 10mM basic sodium phosphate, 2mM monobasic potassium phosphate, pH 7.4 with 0.05% Tween-20) or other phosphate or Tris based buffers.

The kit according to the invention preferably further comprises a container with a secondary antibody which recognizes human immunoglobulins and which is conjugated with a detection system, preferably the secondary antibody is anti Human IgG conjugated with the enzyme peroxidase of horseradish.

The kit according to the invention preferably further comprises a container with the substrate for the detection preferably TMB (3,3â € ², 5,5â € ²-tetramethylbenzidine) in the case in which peroxidase is used.

The kit according to the invention preferably further comprises a container with a solution for blocking the detection reaction, preferably sulfuric acid in the case of TMB, preferably at a concentration of 2M.

The scientific discovery underlying the present invention emerged in the course of a study which had as its objective the identification in the cerebrospinal fluid and serum of subjects affected by multiple sclerosis of autoantibodies capable of recognizing circulating autoantigens present in the same CSF. .

The first experiments were aimed at defining the cerebrospinal CSF protein expression profile of subjects with multiple sclerosis and the comparison with the serum proteomic map confirmed that most CSF proteins are serum derived. Furthermore, the results obtained show that the removal of albumin and immunoglobulins from the CSF samples before subjecting them to two-dimensional electrophoresis allows to obtain a map that well represents the secretome of the central nervous system, as the less abundant proteins are also present.

To evaluate the presence of autoreactive antibodies in the cerebrospinal fluid of patients with multiple sclerosis, an immunoproteomic approach was used, which combines the resolving power of two-dimensional electrophoresis with the specificity of the antigen-antibody reaction and thus allows to identify the signals immunoreactive proteins. It made it possible to identify an autoantigen, transferrin, which is involved in the metabolism of iron. The clinical significance of the reduction of iron within the central nervous system is known and the presence of anti-transferrin autoantibodies in the CSF of subjects with multiple sclerosis could contribute to the demyelination of neurons and therefore to the immunopathogenesis of the disease.

Autoreactive anti-transferrin antibodies have also been detected in the serum of patients and, to a lesser extent, in the serum of healthy individuals where they probably contribute to the mechanisms of the so-called â € œinborn autoimmunityâ €. However, the significant difference in concentration detected between the serum samples of individuals with multiple sclerosis and healthy individuals can be used to discriminate the presence of the disease.

From these experimental evidences it was therefore possible to develop an enzyme immunoassay, based on the ELISA method, which allows the identification and quantization of serum anti-transferrin antibodies for the diagnosis of multiple sclerosis with a non-invasive test.

The present invention can be better understood in the light of the experimental activity carried out and described below.

EXPERIMENTAL PART

Proteomic profile of cerebrospinal fluid in multiple sclerosis

A proteomic approach was used to define the protein expression profile of the cerebrospinal fluid of subjects suffering from multiple sclerosis: the CSF samples collected and prepared as described later in Materials and Methods, were subjected to 2D-IPG / 2D two-dimensional electrophoresis. SDS-PAGE and analyzed as described later in Materials and Methods.

Figure 1A shows a representative gel, where the CSF proteins of an individual with multiple sclerosis have been separated, showing the identified proteins.

To highlight the intrathecal synthesized proteins, ie those synthesized within the central nervous system, the proteomic map of the cerebrospinal fluid was compared with that of the serum of the same subjects (figure 1B). The comparative analysis of the maps obtained showed, in accordance with the data reported in the literature, that the protein profile of the liquor sample is largely overlapping with that of the corresponding serum samples. In fact it is known that almost 80% of the proteins present in the liquor are of serum derivation and among these above all albumin and immunoglobulins, which represent approximately 50% and 15% of the liquor peptides respectively. Instead, some proteins and certain protein isoforms are preferentially or exclusively present in the cerebrospinal fluid (indicated with the oval in figure 1A): this indicates their synthesis within the central nervous system. It is precisely on these proteins that it is appropriate to turn attention to identify potential molecular markers of multiple sclerosis.

In order to increase the resolution of less abundant intrathecal synthesis proteins and to obtain a characterization of the CSF protein expression profile that was as complete and representative as possible, the removal of albumin and class G immunoglobulins was performed (IgG) from samples prior to two-dimensional electrophoresis as described later in Materials and Methods.

These, in fact, are the most abundant proteins in the cerebrospinal fluid and their presence can mask other less represented protein spots. Furthermore, to further increase the resolution of the proteins present in low concentration, CSF samples from different patients (with similar clinical characteristics) were pooled to form four mixtures (pool I, II, III and pool IV) as described later in Materials and methods.

Therefore, the CSF pools of subjects affected by multiple sclerosis, after the removal of albumin and IgG, were subjected to 2D-IPG / SDS-PAGE electrophoretic analysis and the proteomic maps obtained (Figure 2B shows a representative gel ) were compared with those of CSF obtained previously (Figure 2A). The comparative analysis highlighted the effective elimination of a large part of albumin and IgG (inside the rectangles in figures 2A and 2B), the persistence of both other proteins of serum origin and those characteristic of liquor (these the latter delimited by an oval in Figures 2A and 2B) and the appearance of some protein spots (highlighted with a square in Figure 2B) not previously displayed, because they correspond to the less abundant proteins.

In this way a proteomic map was obtained which can be referred to as the secretome of the central nervous system and which was used for the subsequent immunoproteomic study.

Identification of autoreactive antibodies in the cerebrospinal fluid

An immunoproteomic approach was used to identify any reactive antibodies to proteins present in the cerebrospinal fluid of subjects suffering from multiple sclerosis: three of the four available liquor pools, after the removal of albumin and IgG, were subjected to two-dimensional electrophoresis and analyzed by western blot for the detection of autoantibodies present in the same biological fluid as described later in Materials and Methods.

In particular, each of the three two-dimensional maps where the proteins of the cerebrospinal CSF pools were separated after albumin and IgG depletion was hybridized with the corresponding non-depleted CSF pool, in order to identify the presence of any autoreactive IgG ( autoantibodies) that recognize proteins of the same fluid. In the autoradiographic plates obtained, the appearance of immunoreactive signals was observed in all three cases (figures 3A, 3B, 3C); the corresponding protein spots (figures 3D, 3E, 3F) were analyzed by gel matching with the previously created CSF reference map and thus were identified with some isoforms of the transferrin protein. The detected anti-transferrin autoantibodies could have potential application as molecular markers for the study and / or diagnosis of multiple sclerosis.

The interest in the anti-transferrin antibody as a possible biomarker for the disease is supported by the further observation that it appears to be absent in the cerebrospinal fluid of unaffected subjects, as emerged from the checks carried out using Western methodologies. blotting as described later in Materials and Methods, hybridizing the depleted pools of multiple sclerosis with the non-depleted CSF of individuals with migraine, where no immunoreactive spots are observed (Figures 4A and 4B); this suggests that the presence of antitransferrin autoantibodies may be specifically related to multiple sclerosis.

To verify the correspondence of the immunoreactive spots with transferrin, two-dimensional western blots were performed, as described later in Materials and Methods, using a commercial preparation of transferrin purified from human plasma as antigen.

Figure 5A shows the proteomic map of the preparation used, where in addition to transferrin there are also other plasma proteins not completely removed during the purification process. As confirmation of the transferrin enrichment, the map obtained was hybridized, as described later in Materials and Methods, with the specific anti-transferrin antibody available on the market, which recognized both the canonical isoforms of transferrin and some not canonicals never described in literature (indicated by numbers in figure 5B). The purified transferrin map was then hybridized, as described later in Materials and Methods, with the non-depleted cerebrospinal fluid of pools I, II or III and immunoreactive signals were obtained in correspondence with protein spots which, by means of gel matching with the analytical map obtained previously, they were identified with some transferrin isoforms (a representative image is shown in figure 5C). In the control carried out by hybridizing the map in question with the CSF of subjects suffering from migraine no reaction was observed (data not shown). These observations demonstrate that the spots initially identified as transferrin by gel matching with the CSF reference map actually correspond to some isoforms of this protein.

To confirm the specificity of the binding of autoreactive IgG present in the CSF of subjects with multiple sclerosis with transferrin, a competition assay was conducted as described later in Materials and Methods. It consists in incubating the sample containing the antibodies with the antigen potential and after testing its immunoreactive capacity: in the presence of a specific binding, as the antigen concentration increases, the binding sites in the antibodies are saturated and the their immunoreactivity.

Therefore, non-depleted cerebrospinal fluid from pool I, II or III was incubated with the transferrin preparation and then used to hybridize the proteomic map of the same purified protein. In the case of a 3-hour incubation with 50Î1⁄4g / ml of transferrin, an inhibition of about 50% of the binding capacity of self-reactive IgG was observed (figure 6B compared to 6A), while with a 5 hours incubation with 200Î1⁄4g / ml of protein the immunological reaction was almost totally inhibited (figure 6C). These observations confirm that the link between the autoantibodies present in the cerebrospinal fluid of subjects suffering from multiple sclerosis and transferrin is specific and can be inhibited in a dose- and time-dependent manner by incubating the sample in question with the purified antigen.

These results therefore suggest that autoantibodies that recognize and bind transferrin are present in the cerebrospinal fluid of subjects with multiple sclerosis. This discovery takes on particular significance in light of the fact that this protein could have a biological role in the pathogenesis of the disease. In fact, transferrin is responsible for the transport of iron in the central nervous system through the blood brain barrier and is involved in its homeostasis. Iron is an essential factor for the development of oligodendrocytes and for the production of myelin, therefore a deregulation of these mechanisms could lead to the onset of neurodegenerative diseases. Furthermore, transferrin has been directly correlated to the process of myelination and in the brain of individuals with multiple sclerosis its distribution is altered; recently a study also showed a decrease in transferrin levels in the cerebrospinal fluid of these subjects. Therefore, the anomalous presence of anti-transferrin autoantibodies in the CSF could sequester the protein, reduce the amount circulating in the central nervous system and make it unavailable for binding with iron, thus contributing to the demyelination responsible for the disease.

The autoreactive antibodies against transferrin present in the cerebrospinal fluid of subjects with multiple sclerosis could derive from intrathecal synthesis or originate from the blood, following the compromise of the blood brain barrier caused by the inflammatory condition that characterizes the disease. In both cases, anti-transferrin autoantibodies could also be present in the bloodstream of these individuals: for this reason their existence in serum samples was also investigated. In the field of clinical proteomics, in fact, it is widely recognized that the identification of molecular markers in a biological sample such as serum provides a significant diagnostic advantage, since the collection of a blood sample requires procedures considered to be minimally invasive. . Furthermore, from the experimental point of view, the use of serum for the analyzes allows to have available also healthy control samples or those coming from subjects affected by other pathologies.

Identification of autoreactive antibodies in serum

For the identification of anti-transferrin antibodies in the serum of subjects suffering from multiple sclerosis, proteomic maps of the cerebrospinal fluid pools (after removal of albumin and IgG) hybridized with the respective serum pools or with single samples were used as described in the previous section and in the Materials and Methods.

Immunoreactive spots corresponding to transferrin isoforms were observed in all cases.

Figure 7 shows the two-dimensional western blots performed by hybridizing the pool IV map of CSF samples from patients with multiple sclerosis, depleted of albumin and IgG, with the corresponding serum pool (Figure 7A) or with a single serum sample. of a sick individual (figure 7B): various isoforms of the target antigen have been recognized, both by the antibodies present in the pool and in the single sample, in which they are evidently in a sufficiently high concentration.

Also in this case, to verify the correspondence of the immunoreactive spots with the transferrin, two-dimensional western blots were carried out using the commercially available preparation of transferrin purified from human plasma as antigen.

Figure 8 shows the two-dimensional western blots performed by hybridizing the purified transferrin map with the serum of two sick subjects (Figures 8A and 8B): in both cases the canonical isoforms of transferrin were recognized and in one of the two also some of those non-canonical (Figure 8A).

These results indicate that autoreactive antibodies directed against transferrin are also present in the serum of subjects with multiple sclerosis. However, before it could be stated with certainty that serum anti-transferrin antibodies can be used as molecular markers of the disease, further analyzes were needed on serum samples taken from patients and unaffected individuals, in order to confirm their presence. in the former and their absence in the latter.

To make the detection system easier, faster and applicable to a large number of samples, a one-dimensional western blot assay based on SDS-PAGE was used as described later in Materials and Methods.

Furthermore, since the resolving power of the gels used in the protein fractionation on one dimension is lower than that of the two-dimensional gels, in order to reduce the risk of false-positive signals due to the presence of the other proteins in the purified transferrin preparation, it was used as the antigen is the commercial protein produced by recombinant DNA technology, which has a purity of essentially 100% and is commercially available. Figure 9 shows a comparison between the immunoreactive signals generated by hybridization of recombinant transferrin with representative serum samples from a subject with multiple sclerosis (Figure 9B) and a healthy individual (Figure 9C); as a control of the specificity of the binding, the signal generated by the previously described recombinant transferrin hybridized with the anti-transferrin antibody available on the market is also reported (figure 9A).

These data confirm that in the serum of sick subjects there are autoreactive antibodies against transferrin, which are therefore able to recognize and bind both the antigen present in the cerebrospinal fluid of patients, that purified from human plasma, and the recombinant one. The weak positive signal observed using samples from healthy subjects suggests that antitransferrin antibodies are present, with some frequency and in small quantities, even in the serum of individuals not affected by multiple sclerosis.

The presence of autoreactive antibodies in an organism is generally considered a failure of the mechanisms that regulate immune tolerance and the cause of the onset of autoimmune diseases. In fact, although these autoantibodies are a distinctive feature of these pathologies, it has been seen that they can also be present in healthy individuals where they could perform the functions of homeostasis of the tissues, cleaning them from metabolic waste and from the elements that have finished their function. Recently, Hedegaard's research team demonstrated the presence of immunoreactive antibodies against myelin basic protein (MBP), believed to be one of the causes of demyelination in multiple sclerosis, in the serum of both affected and healthy individuals. It seems, therefore, that autoantibodies are also present in healthy subjects, so much so that we speak of â € œinborn autoimmunityâ €; it is therefore believed that external factors intervene to trigger autoimmune diseases that contribute to altering the delicate balance of their concentrations, thus exacerbating the response against the bodyâ € ™ s own antigens.

In light of these considerations, the significant difference in anti-transferrin antibody concentration detected between the serum samples of individuals with multiple sclerosis and healthy individuals may be sufficient to discriminate the presence of the disease. This important observation supports the idea of a possible use of anti-transferrin autoantibodies as molecular markers of multiple sclerosis; setting up a system for their detection in serum could provide a powerful diagnostic tool to help clinicians work. Since the one-dimensional western blot is not the most suitable method for detecting changes in the concentration of autoantibodies in serum, a more sensitive method such as the ELISA enzyme immunoassay has been developed.

ELISA assay

The purpose of a laboratory test in the field of autoimmunity is to determine in a sensitive and specific way the autoreactive antibodies directed against a given antigen, typically present in blood samples, so as to provide in the reporting of analytical results a diagnostic value on their concentration. Among the different methods designed to detect specific soluble molecules in plasma, the enzyme immunoassays are those that more than others guarantee characteristics of simplicity and speed of execution, reliability in quantitative estimates, cost-effectiveness. The peculiarity of enzyme immunoassays is to exploit in a coupled way an immunological reaction, such as the antigen-antibody bond, to selectively bind the researched molecule and an enzymatic reaction to produce a colored signal that can be easily measured quantitatively with special photometers. The immunological component of the assay guarantees its specificity, while the enzymatic component ensures its analytical sensitivity.

In particular, the ELISA, acronym of the English expression Enzyme-Linked ImmunoSorbent Assay (immuno-absorbent assay linked to an enzyme) is a versatile method of immunoassay that is widely used to detect biomarkers in various pathologies. To identify a particular antibody possibly present in a sample, the specific antigen against which it is directed is used: the quantity of the antigen-antibody complex formed, made visible by means of a colorimetric reaction, is an indication of the concentration of the antibody that you want to evaluate in the sample under examination.

To detect the presence of anti-transferrin antibodies in serum samples on a large scale, an ELISA assay was performed, as described in detail in Materials and Methods, in which the transferrin antigen is immobilized on a microtiter plate, which will bind the specific autoantibodies possibly present in the serum. In order to ensure the specificity of the antigen-antibody interaction, it is preferable to use recombinant transferrin, which is free from contamination of other proteins; the results that have been obtained with the one-dimensional western blots show that the one that has been used is recognized by the anti-transferrin autoantibodies of the serum and therefore can be used for this purpose. Therefore, it is possible to imagine the development of a diagnostic kit, based on ELISA technology, which allows to detect variations in the concentration of anti-transferrin autoantibodies in the serum of subjects suffering from multiple sclerosis compared to healthy individuals.

The experiments carried out for the development of the assay allowed to identify the suitable experimental conditions to highlight the differences in the quantity of anti-transferrin autoantibodies present in the serum of subjects suffering from multiple sclerosis compared to healthy individuals used as controls. Figure 10 shows the graph relating to an assay in which it is observed that, using recombinant transferrin at a concentration of 3.2 Î1⁄4g / ml as antigen to be adsorbed in the wells of the microtiter plate, which corresponds to 0.5 Î1⁄ 4g / cm <2>, we are able to discriminate the different quantity of anti-transferrin autoantibodies present in the serum of a sick subject compared to a control. The experiments were also conducted using transferrin purified from human plasma, which has a purity of 98%, as antigen to be adsorbed in the wells of the plate, and the same results were obtained. On the other hand, by using a protein other than transferrin, such as BSA (bovine serum albumin) as antigen to be adsorbed in the wells, there is no colorimetric reaction and this demonstrates the specificity of the interaction of the anti-transferrin autoantibodies present in the serum with transferrin. adsorbed.

In order to evaluate the sensitivity and specificity of the assay of the diagnostic method according to the invention, as well as to establish the threshold value of the anti-transferrin concentration to be associated with the disease, in addition to the serum of affected individuals and healthy ones, including samples from patients with autoimmune diseases and with different neurological diseases. The results of the sera from 149 individuals tested, grouped into 4 classes, are shown in figure 11.

The results show that the assay is highly specific.

MATERIALS AND METHODS

CSF and serum samples

Cerebrospinal fluid and serum samples from individual patients or mixtures of samples (referred to as pools) were used for analysis.

The four CSF pools were prepared by mixing samples taken from subjects with relapsing-remitting multiple sclerosis (MS-RR), heterogeneous from an age point of view, but with similar clinical characteristics (table 1). In particular, pool I consists of 3 samples, the II from 7, the III from 9 and the IV from 8.

Table 1. Demographic and clinical characteristics of the patients from whom the samples were obtained.

Champion Gender / Age Diagnosis Status al Bande Steroids

sampling oligoclonal sampling

1 F / 26 SM-RR 0 0 multiple Pool I 2 F / 26 SM-RR 0 0 multiple Pool I 3 F / 21 SM-RR 0 0 multiple Pool I 4 F / 41 SM-RR 0 0 multiple Pool II 5 F / 56 SM-RR 0 0 multiple Pool II 6 M / 33 SM-RR 0 0 none Pool II 7 M / 32 SM-RR 0 0 none Pool II 8 F / 29 SM-RR 0 0 none Pool II 9 M / 41 SM-RR 0 0 none Pool II 10 F / 35 SM-RR 0 0 none Pool II 11 F / 25 SM-RR 0 0 multiple Pool III 12 F / 23 SM-RR 0 0 none Pool III 13 F / 39 SM- RR 0 0 multiple Pool III 14 M / 35 SM-RR 0 0 not performed Pool III 15 M / 37 SM-RR 0 0 not performed Pool III 16 F / 37 SM-RR 0 0 multiple Pool III 17 F / 53 SM- RR 0 0 none Pool III 18 M / 30 SM-RR 0 0 multiple Pool III 19 M / 35 SM-RR 0 0 multiple Pool III 20 F / 49 SM-RR 0 0 none Pool IV 21 M / 22 SM-RR 0 0 none Pool IV 22 M / 24 SM-RR 0 0 multiple Pool IV 23 F / 30 SM-RR 0 0 none Pool IV 24 F / 56 SM-RR 0 0 multiple Pool IV 25 F / 49 SM-RR 0 0 multiple Pool IV 26 F / 38 SM-RR 0 0 multiple Pool IV 27 F / 32 SM-RR 0 0 multiple Pool IV

CSF samples from migraine sufferers were used as a control.

The CSF samples, taken by lumbar puncture, were centrifuged at 1,000g for 10 minutes (at 4 ° C), in order to eliminate the insoluble material and any cellular component present; the supernatant obtained was subjected to dialysis (at 4 ° C), to remove excess salts, and then lyophilized to concentrate the proteins present. Then the samples were resuspended in the ISOT solubilization buffer (8M urea, 4% CHAPS, 40mM Tris, 65mM DTE), necessary for the subsequent two-dimensional analyzes, and stored at â € “80 ° C until their use.

The corresponding patient serum was provided for each CSF sample, and serum samples from individuals without any pathology were also made available.

Serum samples for two-dimensional analyzes were directly diluted 1:10 in the ISOT buffer.

Protein concentration in CSF and serum samples was determined by the Bradford assay (Bradford, 1976).

2D-IPG / SDS-PAGE two-dimensional electrophoresis

Isoelectric focusing (IPG), for the separation of proteins in the first dimension, was performed on strips of acrylamide / bisacrylamide gel with a non-linear gradient of pH 3-10 (GE Healthcare, USA). The strips, 180mm long and 3mm wide, were rehydrated (1 hour at 0V at 20 ° C and 10h at 50V) in a solution containing 8M urea, 2% CHAPS, 10mM DTE, 0.5% of the ampholyte mixture (GE Healthcare, USA) and traces of bromophenol blue. From time to time, the following sample aliquots, previously solubilized in ISOT, were added to this solution:

ƒ 60Î1⁄4g of cerebrospinal fluid protein from a patient with multiple sclerosis, for the analytical gels to be silver-stained;

ƒ 60Î1⁄4g of serum proteins from patients with multiple sclerosis, for the analytical gels to be subjected to silver coloring;

ƒ 30Î1⁄4g of proteins from the cerebrospinal fluid pool after removal of albumin and IgG, for the analytical gels to be silver-stained; ƒ 60Î1⁄4g of proteins from the cerebrospinal fluid pool after removal of albumin and IgG, for immunoproteomic analysis by western blot; ƒ 30Î1⁄4g of transferrin purified from human plasma, for the analytical gels to be subjected to silver coloring and for the immunoproteomic analysis.

Protein separation was achieved (at 20 ° C) by subjecting the system to a linear voltage increase from 200V to 3,000V during the first 4.5 hours and then constant at 8,000V for 8 hours. The strips were then equilibrated for 30 minutes in a solution containing 6M urea, 30% glycerol, 2% SDS, 2% DTE, 50mM Tris-HCl at pH 6.8 and then for 20 minutes in a similar solution, where the DTE It was replaced with 2.5% iodoacetamide and bromophenol blue dye added.

In the second dimension (SDS-PAGE) the proteins present in the strips were separated, by electrophoresis on a vertical support of 18x20 cm, with a linear gradient 9-16% of polyacrylamide (acrylamide: PDA 30: 0.8, 0.025% thiosulfate of sodium, 0.056% APS, 0.075% TEMED, 375mM Tris-HCl at pH 8.8). The run was performed at a constant 14W / gel at 10ºC, until the reference dye reached the bottom edge of the gel. The sliding buffer used is made up of 25mM Tris, 192mM Glycine, 0.1% SDS.

At the end of the two-dimensional electrophoresis, for the visualization of the protein spots, the gels were subjected to staining with silver nitrate or, in the case of immunoproteomic analysis by western blot, the proteins were transferred to a nitrocellulose membrane.

Acquisition of two-dimensional maps and analysis

For the proteomic analysis the maps were acquired with the ImageScanner II densitometer (GE Healthcare, USA) and the images were qualitatively and quantitatively analyzed with the ImageMaster 2D Platinum 6.0 software (GE Healthcare, USA), which allows the identification of the protein spots, their volumetric comparison and the overlap between different gels (gel matching).

In order to attribute the correct coordinates of isoelectric point and molecular weight to the spots displayed on the two-dimensional maps, a calibration was performed using some serum proteins as a reference.

In the proteomic map of the cerebrospinal fluid, the spots corresponding to the specific proteins of this sample were identified by gel matching with a reference map present in the SWISS-2DPAGE database.

Removal of albumin and IgG from CSF samples

To increase the sensitivity and resolving power of the two-dimensional analyzes, the CSF samples were subjected to the removal of albumin and IgG immunoglobulins using the â € œAlbumin and IgG Depletion SpinTrapâ € kit (GE Healthcare, USA). It is based on the use of affinity columns where a mixture of agarose beads has been packed, on which the highly specific ligand for albumin and IgG has been immobilized.

The portion of liquor to be subjected to depletion, both for single samples and for pools, was dialyzed, lyophilized, resuspended in a suitable binding buffer (150mM sodium chloride, 20mM sodium phosphate at pH 7.4) and loaded on the affinity column. The eluate was collected by centrifugation and, after dialysis and lyophilization, was resuspended in the ISOT buffer and quantized.

Western Blot

To detect the immunoreactive signals, after the electrophoretic run, the gel-separated proteins were transferred onto a nitrocellulose membrane in order to be subjected to western blot analysis. In particular, the analyzed samples were:

ƒ the proteins of the cerebrospinal fluid pools depleted of albumin and IgG, after two-dimensional fractionation;

ƒ transferrin purified from human plasma, after two-dimensional fractionation;

ƒ recombinant transferrin, after one-dimensional fractionation.

The transfer was carried out by superimposing the membrane (Hybond-ECL; GE Healthcare, USA) on the gel, on which the CSF or transferrin samples were fractionated, and subjecting the complex to a constant electric field of 50V for a hour (at 4 ° C), in a suitable transfer buffer (25mM Tris, 192mM glycine, 20% methanol). Then the membrane was colored with a solution of Ponceau red (0.2% Ponceau S in 3% trichloroacetic acid), in order to verify the transfer of the proteins and to be able to mark the position of some of them, from use later as a reference when superimposing it with the corresponding map colored with silver salts. Subsequently the membrane was incubated for one hour with a solution of 5% bovine serum albumin (BSA) in a TBS-T buffer (20mM Tris, 140mM sodium chloride, 0.1% Tween-20), in order to to saturate the residual active sites present on the nitrocellulose. After saturation, the membrane was incubated for 14-16 hours at 4 ° C with the primary antibody in TBS-T buffer. In particular, the membranes on which the proteins of the albumin and IgG depleted cerebrospinal liquor pools were transferred were incubated:

ƒ with the respective non-depleted CSF pool, diluted 1: 100 in TBS-T;

ƒ with the liquor of subjects with migraine, used as a negative control, diluted 1: 100 in TBS-T;

ƒ with the corresponding serum pool, diluted 1: 10,000 in TBS-T;

ƒ with serum from patients with multiple sclerosis, diluted 1: 10,000 in TBS-T.

While the membrane on which the purified transferrin from human plasma was transferred was incubated:

ƒ with non-depleted liquor pools, diluted 1: 100 in TBS-T;

ƒ with the serum of subjects with multiple sclerosis, diluted 1: 10,000 in TBS-T; ƒ with the specific polyclonal anti-transferrin antibody available on the market (Santa Cruz Biotechnology, USA), diluted 1: 1,000 in TBS-T. Finally, the membrane on which the recombinant transferrin was transferred was incubated:

ƒ with the serum of subjects with multiple sclerosis, diluted 1: 10,000 in TBS-T; ƒ with the serum of healthy individuals, diluted 1: 10,000 in TBS-T;

ƒ with the specific anti-transferrin polyclonal antibody, diluted 1: 1,000 in TBS-T.

After incubation with the primary antibody, the membrane was washed in TBS-T to remove the unbound portion and then it was incubated for one hour with the secondary antibody â € œanti-human-IgGâ € (GE Healthcare, USA), to detect the autoantibodies present in the CSF and serum, or â € œanti-rabbit-IgGâ € (Santa Cruz Biotechnology, USA), to detect the anti-transferrin which It was generated in rabbits diluted 1: 20,000 and 1: 10,000 in TBS-T, respectively. Then the membrane was subjected to further washes in TBS-T to remove the unbound secondary antibody molecules and then exposed to chemoluminescent immunological detection using the Super Signal West Pico Chemioluminescent Substrate kit (Thermo Scientific, USA). In fact, the secondary antibodies are conjugated to the horseradish peroxidase enzyme which, in the presence of its specific substrate, develops a chemioluminescence reaction capable of impressing an autoradiographic plate and giving immunoreactive signals in correspondence with the spots (in the two-dimensional maps) or protein bands (in one-dimensional gels).

Competition essay

The antibody competition assay was performed by incubating the CSF pool (containing albumin and IgG) diluted 1: 100 in TBS-T, for 3 hours or for 5 hours with 50Î1⁄4g / ml or 200Î1⁄4g / ml of the purified transferrin, before using it to hybridize the proteomic map by western blot. In this way the transferrin saturates the specific binding sites of the anti-transferrin antibody which, therefore, is no longer reactive towards the antigen present in the gel with which it will be hybridized and will not give an immunological reaction.

One-dimensional electrophoresis

The fractionation of recombinant transferrin by one-dimensional electrophoresis (SDS-PAGE) was carried out in a polyacrylamide gel in the presence of SDS (8% acrylamide: bisacrylamide 30: 0.8, 0.1% SDS, 0.1% APS, 0.075% TEMED, 375mM Tris-HCl at pH 8.8). 0.5Î1⁄4g of transferrin for each well was loaded onto the gel. The sliding buffer used for the electrophoretic stroke, performed at a constant voltage of 150V, is composed of 25mM Tris, 192mM Glycine, 0.1% SDS.

At the end of the electrophoresis, the proteins were transferred to a nitrocellulose membrane for western blots.

Preparations of the transferrin protein

Two preparations of transferrin were used, both marketed by Sigma-Aldrich (USA): one obtained by purification of the protein from human plasma and one produced by recombinant DNA technology. For the two-dimensional electrophoresis the samples were diluted in ISOT.

ELISA assay

The ELISA enzyme immunoassay was carried out in transparent microtiter plates (96 wells) in polystyrene with â € œhigh-bindâ € and â € œeasywashâ € characteristics (Corning, USA), in order to favor the binding of the antigen and facilitate the washing steps. To adsorb the antigen in the wells of the plate, 50Î1⁄4l aliquots of a solution of recombinant transferrin (Sigma-Aldrich, USA) or bovine serum albumin (BSA) at a concentration of 3.2Î1⁄4g / ml in a carbonate buffer 0.1M sodium bicarbonate at pH 9.4 were incubated overnight at 4 ° C. The amount of unbound antigen was removed by 3 washes with 300Î1⁄4l of a PBS-T buffer (137mM sodium chloride, 2.7mM potassium chloride, 10mM basic sodium phosphate, 2mM monobasic potassium phosphate, pH 7.4 with 0.05% Tween-20). Subsequently the plate was incubated for one hour at room temperature with 300Î1⁄4l of a 2% BSA solution in the PBS-T buffer, in order to saturate the residual active sites present on the polystyrene. Then, the plate was incubated for 2 hours at room temperature with 50Î1⁄4l of the serum to be analyzed diluted 1: 100, 1:50 or 1:20 in the 2% BSA solution in PBS-T. After carrying out 5 washes with 300Î1⁄4l of PBS / T, 50Î1⁄4l of horseradish peroxidase conjugated anti-human-IgG secondary antibody (Dako, Denmark) diluted 1: 5000 in BSA 2 solution was added to each well. % in PBS-T and incubated for one hour at room temperature. The quantity of unbound secondary antibody was removed by 5 washes with 300Î1⁄4l of PBS / T and subsequently the colorimetric detection was performed by adding to each well 100Î1⁄4l of TMB peroxidase substrate (Thermo Scientific, USA) which It was incubated for 15-20 minutes at room temperature away from light. Then, the colorimetric reaction was stopped with 100Î1⁄4l of 2M sulfuric acid and the absorbance at 450nm was measured by a plate reader.

Reagents

All reagents used, unless expressly indicated, were purchased from Sigma-Aldrich (USA).

Claims (10)

CLAIMS 1. A method for the diagnosis and / or prognosis of multiple sclerosis and / or for the monitoring of therapy, said method comprising the identification and quantification of anti-transferrin antibodies in a biological fluid. 2. Method according to claim 1 wherein said biological fluid is selected from serum or cerebrospinal fluid. Method according to any one of claims 1-2 in which the determination of the presence of anti-transferrin antibodies is carried out by means of the ELISA method. 4. Method according to claim 3 wherein the anti-human-IgG secondary antibody conjugated with horseradish peroxidase is used for the detection of the antigen-antibody reactions. 5. Diagnostic kit for multiple sclerosis for the method according to any one of claims 1-4, said kit comprises at least one microtiter plate on which transferrin has been immobilized 6. A kit according to claim 5 wherein the immobilized transferrin has a purity higher than 97% by weight. 7. Kit according to any one of claims 5-6 wherein the immobilized transferrin is at a concentration of 1.0-20 Î1⁄4g / ml. 8. Kit according to claim 6 wherein the immobilized transferrin is at a concentration of 2.0-5 Î1⁄4g / ml. 9. Kit according to any one of claims 5-8 further comprising: ∠’at least one container containing an anti-transferrin antibody sample to be used as a positive control; ∠’at least one container containing a sample of a molecule that can act as a negative control; ∠’at least one container containing a secondary antibody that recognizes human immunoglobulins and that is conjugated with a detection system; ∠’at least one container with the phosphate or tris base buffer solution; ∠’at least one container containing a substrate for the detection system. ∠’at least one container with the solution to stop the detection reaction. 10. Kit according to any one of claims 5-9 wherein: ∠’the negative control is bovine serum albumin; ∠’the buffer solution is PBS-T; ∠’secondary antibody is anti human IgG conjugated to horseradish peroxidase; ∠’the substrate of the peroxidase is 3,3â € ², 5,5â € ²-tetramethylbenzidine (TMB); ∠’The stop solution is a 2M sulfuric acid solution