The invention relates to a method for detecting and supervising neurodegenerative diseases, which consists in detecting the presence of antibodies of the A-isotype and/or M-isotype which are directed against the antigens which are associated with these diseases. The invention also relates to a kit for the implementation of this method.

This application is a National Stage application under 35 USC 371 of PCT/FR02/00927, with an international filing date of Mar. 15, 2002.

A subject of the present invention is a method and a kit for the supervision of neurodegenerative diseases.

The invention can notably be applied in the medical field and in the immunological field.

Despite the immense medical progress over the last fifty years, a certain number of diseases, which are known for a long time or which are of recent appearance, remain strictly speaking incurable, despite a significant cost for Public Health. Neurodegenerative diseases may in particular be cited, such as Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, auto-immune diseases, hepatites, degenerative and inflammatory rheumatisms including rheumatoid arthritis (RA).

In these diseases, the nosological diagnosis is given, certain mechanisms and stages of the lesion process are known, symptomatic treatments are given, which are often very expensive, but the results are not at the height of expectations.

Multiple sclerosis is a multilocular demyelinising pathology of the central nervous system which affects the white matter of the brain and of the spinal cord, and which is a very highly disabling pathology of insidious and unpredictable progression.

In France, MS affects 1 individual per 1000 in full maturity (40 to 60 cases for 100,000 inhabitants), i.e. 50,000 French people, and 2,000 new cases are to be counted every year. 2 million cases are counted in the world.

The disease begins in the young adult, between 15 and 50 years old (between 20 and 40 years old in particular). Its incidence is twice as much in the woman as in the man.

MS has three distinct clinical forms: remittent, progressive and remittent progressive.

Amyotrophic lateral sclerosis (ALS) or Charcot's disease is a neurological disease of rapid progression and of unknown aetiology. The disease is characterised by a degenerative attack of the motor neurones of the brain, of the cerebral trunk and of the anterior horn of the spinal cord, whereas the sensory neurones are not affected and whereas the intellectual functions remain intact, leaving the patients fully conscious of the progression of their disease and of the deterioration of their functional capacities.

The prevalence of ALS is equal to that of MS. It is five times higher than that of Huntington's disease. More ill people die every year from ALS than from MS or Huntington's disease.

ALS occurs most often between 45 and 70 years old, affecting 1.5 to 2 times more men than women. The disease concerns subjects which are younger and younger: cases exist having early beginning before 40 years old.

Its incidence, i.e. the number of new cases each year, has a tendency to increase in the world for an unknown reason. In France, there would be 5 to 10,000 persons affected, and about 1,100 new cases appear every year.

The progression of this disease is dramatic, and is always fatal. It proceeds towards a progressive aggravation, with an installation of a bedridden state and of a respiratory insufficiency through attack of the intercostal and diaphragm musculature, which rapidly leads to death (in 3 to 5 years).

ALS has a progressive clinical heterogeneity which leads a biological heterogeneity to be considered.

Rheumatoid arthritis (RA), formerly called atrophic arthritis, is a progressive chronic polyarthritic inflammatory disease which is responsible for osteocartilaginous damage, which leads to a painful functional impotence through deformation and ankylosis.

It is therefore a potentially severe disease which causes a significant handicap in over half the patients, 10 years after the beginning of the symptoms, and reduces their life expectancy by several years.

Its prevalence in France would be about 1%: it is the most frequent chronic inflammatory rheumatism. It affects 7,000 new individuals per annum, affecting more often women who are still young.

Spondylarthritis (SAR) is a chronic rheumatic disease which manifests itself by a lumbosacral attack which leads to a progressive ankylosis. This immunological and inflammatory disease is linked to an over-expression of the histocompatibility antigen B27 (found in 90% of cases).

The significance of these diseases both from the point of view of the number of persons affected, and the cost to Public Health, is therefore not negligible.

Hitherto, no biological test exists for evaluating the stage and/or for foreseeing the progression of these diseases. Only a clinical examination, optionally completed by additional examinations, enables the diagnosis of these diseases to be made. A real need therefore does exist to be able to have a specific progressive diagnostic test at one's disposal which can palliate the insufficiencies of the clinic.

The Applicant has demonstrated that the occurrence of neurodegenerative diseases was associated with antigen modifications, which are integrated by immunocompetent cells, manifesting itself by the production of circulating antibodies of the A-isotype and/or M-isotype. These antibodies are directed against the antigens indicated in Table 1 (see Original Patent), showing the antigen modifications and the bacterial factors which participate in the chronicity of these diseases:

The detection of these antibodies can therefore enter within the context of a general diagnosis of neurodegenerative diseases, and the supervision of these diseases.

Such a method of detecting these antibodies in a biological fluid can comprise the following steps: placing said fluid in contact with each one of the following antigens: Pal, Ole, Myr, Pl, MDA, Ach, FarCys, Aze, NO-Cys, NO-Tyr, NO-Trp, NO-His, NO2-Tyr, NO-Phe, NO-Arg, NO-Asn, NO-Met, NO-Cr, NO-BSA, Ig3, Ig5, Ig12, Ig13, Ig16, Ig17, Ig19 and revelation of the complexes which are optionally formed between said antigens and the corresponding antibodies.

As is indicated in Table 1, when the presence is detected of an antibody directed against a bacterial antigen "Ig", this antibody can be of the A-isotype and/or of the M-isotype.

Non-bacterial antigens other than bovine serum albumin, which is optionally nitrosylated, are advantageously conjugated.

The term "conjugated antigen" is understood in the sense of the present invention to be an antigen (such as Pal, FarCys, NO-Cys, etc.) which is coupled to a carrier molecule, preferably bovine serum albumin, either directly or via glutaraldehyde or glutaric anhydride. Such a coupling is made in a manner well-known to the person skilled in the art, e.g. as described in Boullerne et al., 1995, 1996; Geffard et al., 1998.

The bacterial antigens are obtained by sonication of cultures of the bacteria indicated in Table 1.

The biological fluid can be, in particular: a serum, a plasma, total blood, urine, cerebrospinal fluid.

The method implemented can advantageously be of the "sandwich" type.

The complexes which are optionally formed can be revealed with the aid of an A or M anti-human immunoglobulin antibody, which is coupled to a label, e.g. a fluorescent label, the biotin/streptavidine system, a (radio)isotopic element, or an enzyme.

The method in accordance with the invention is advantageously carried out with the aid of a suitable solid support.

Any device can be used as a solid support which is adapted to the manipulation of cell suspensions and, preferably, tubes, particular magnetic supports or rigid or flexible microtitration plates of polyethylene, polystyrene, poly(vinyl chloride) or nitrocellulose, comprising microwells.

When a pre-activated microtitration plate is used which has NH2 termini, it is not necessary to "couple" the antigens to a carrier molecule.

The expression "an antibody coupled to a (radio)isotopic element" signifies that the antibody carries, either on an element proper of its structure, e.g. the constituent tyrosine residues, or on an appropriate radical which has been fixed to it, a radioactive isotope enabling it to be determined by counting of the radioactivity which is associated with it.

When the antibody is coupled to an enzyme, this, associated with the use of appropriate reagents, enables a quantitative measurement of this antibody.

The substrate and the reagents are selected so that the final product of the reaction or of the sequence of reactions caused by the enzyme and using these substances be: a coloured or fluorescent substance which diffuses into the liquid medium surrounding the cells and which is the subject of the final spectrophotometric or fluorimetric measurement, respectively, or an insoluble coloured substance which is deposited on the cells and the walls on which they are fixed and which can be the subject, either of a photometric measurement by reflection, or of a visual evaluation, optionally in comparison to a range of standard tints.

When a radio-isotopic element is used, such as 125 iodine for example, the radioactivity associated with the sample is counted in a gamma counter according to any appropriate means.

When an enzyme is used, the appearance of a coloured or fluorescent product is obtained by adding to the solid support a solution which contains the substrate of the enzyme and one or more additional reagents enabling either a coloured product which is soluble in the medium, or an insoluble coloured product, or a soluble fluorescent product, as has been explained above, to be finally obtained. The light signal originating from the thus-treated samples is then measured with the aid of an apparatus which is adapted to each case: a transmission or reflection photometer, or fluorimeter, respectively. When the solid support is a microtitration plate, the reading of the light signal can be made sequentially in all the wells of the same plate by the use of usual automated readers which are common in biology laboratories.

Alkaline phosphatase can be used as enzyme, the preferential substrates of which are para-nitrophenylphosphate for a final spectrophotometric reading, or 4-methyl umbelliferylphosphate for a fluorimetric reading, or 5-bromo-4-chloro-3-indolylphosphate for obtaining an insoluble coloured reaction product. .beta.-galactosidase can similarly be used as enzyme the preferential substrate of which is ortho-nitrophenyl-.beta.-D-galactopyranoside.

Preferably, the anti-Ig antibodies can be conventionally coupled to peroxidase. The reagents used to reveal the peroxidase conjugated to the anti-Ig antibodies contains hydrogen peroxide, which is a substrate of the enzyme, and an appropriate chromogen, e.g. ortho-phenylenediamine, 3,3'-diaminobenzidine or TMB (3,3',5,5'-tetramethylbenzidine) in order to obtain an insoluble final reaction product, or even para-hydroxyphenylpropionic acid in order to obtain a fluorescent reaction product which is soluble in the medium. The colourimetric reaction is stopped with sulphuric acid.

Another preferred embodiment of the invention is the use of anti-immunoglobulin antibodies which are coupled to acetylcholinesterase.

Acetylcholinesterase is coupled to the antibody by preferably using a method which is derived from the one described in French patent No. 2,550,799, or a method which schematically comprises the preparation of fragments of the antibody by a known technique, the modification of the enzyme by reaction with an appropriate heterobifunctional agent, and, finally, the coupling of the products thus obtained.

The revelation of the enzyme activity is preferably made according to the well-known technique which makes use of acetylthiocholine as substrate of the enzyme and Ellman's reagent, or 5,5'-dithio-2-nitrobenzoic acid, as chromogen, according to any variant adapted to the case examined, e.g. the one described in Anal. Chem. 57 (1985) 1170 1173.

The chromogens cited are used as such or as water-soluble salts.

Another subject of the invention is a kit for the implementation of the method described above. This kit advantageously comprises: each one of the following antigens: Pal, Ole, Myr, Pl, MDA, Ach, FarCys, Aze, NO-Cys, NO-Tyr, NO-Trp, NO-His, NO2-Tyr, NO-Phe, NO-Arg, NO-Asn, NO-Met, NO-Cr, NO-BSA, Ig3, Ig5, Ig12, Ig13, Ig16, Ig17 and Ig19; optionally at least one human anti-immunoglobulin antibody, of the A-isotype and/or M-isotype, as defined above.

This kit can also comprise a suitable solid support.

This test notably enables: establishing an early diagnosis of the disease, in particular in persons at risk, and therefore to not create a delay in the treatment of the disease; supervising the progress of the disease, and therefore to be able to adapt the treatment as a consequence.

The detection of the antibodies directed against the following antigens: PI, Ach, Aze, NO-Cys, NO-Tyr, NO-Trp, NO2-Tyr, NO-Asn, NO-Met, Ig5 (A-isotype antibody), Ig12 (A-isotype antibody and M-isotype antibody), Ig13 (A-isotype antibody), Ig16 (A-isotype antibody), Ig17 (M-isotype antibody) and Ig19 (A-isotype antibody), enables in particular the diagnosis of MS to be established. The quantification of the antibodies directed against the following antigens: NO2-Tyr, NO-Tyr, Ach and Ig16 (A-isotype antibody), enables discriminating the particular forms of MS (progressive, remittent, remittent progressive).

Similarly, the detection of the antibodies which are directed against the following antigens: Ole, Pl, MDA, Aze, NO-Cys, FarCys, Ig3 (A-isotype antibody and M-isotype antibody), Ig5 (A-isotype antibody), Ig12 (A-isotype antibody and M-isotype antibody), Ig13 (A-isotype antibody and M-isotype antibody), Ig16 (A-isotype antibody and M-isotype antibody), Ig17 (A-isotype antibody and M-isotype antibody), enables the diagnosis of ALS to be established and, as a function of the antibody titre, to differentiate the 3 classes of this disease.

The detection of the antibodies directed against the following antigens: Pal, Ole, Myr, Pl, MDA, FarCys, Aze, NO-Cys, NO-Phe, Ig3 (A-isotype antibody and M-isotype antibody), Ig5 (A-isotype antibody and M-isotype antibody), Ig12 (A-isotype antibody and M-isotype antibody), Ig13 (M-isotype antibody) and Ig19 (A-isotype antibody), enables the diagnosis of RA to be established.
 

Claim 1 of 27 Claims

1. A method of detecting antibodies which are associated with the occurrence or the progression of Multiple Sclerosis in a biological fluid, which comprises the following steps: placing said fluid in contact with each one of the following antigens: Pl, Ach, Aze, NO-Cys, NO-Tyr, NO-Trp, NO2-Tyr, NO-Asn, NO-Met, Ig5, Ig12, Ig13, Ig16, Ig17 and Ig19, wherein Pl, Ach, Aze, NO-Cys, NO-Tyr, NO-Trp, NO2-Tyr, NO-Asn, NO-Met are non-bacterial antigens and wherein Ig5, Ig12, Ig13, Ig16, Ig17 and Ig19 are bacterial antigens, and revealing the complexes which are formed between said antigens and the corresponding antibodies, wherein said antibodies are of the A-isotype for bacterial antigens Ig5, Ig12, Ig13, Ig16 and Ig19, and of the M-isotype for bacterial antigens Ig12 and Ig17.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.