EP2035832A1 - Procédé de diagnostic et de contrôle de l'évolution de la sclérose en plaques et utilisation d'un kit de test dans ce but - Google Patents

Procédé de diagnostic et de contrôle de l'évolution de la sclérose en plaques et utilisation d'un kit de test dans ce but

Info

Publication number
EP2035832A1
EP2035832A1 EP07817535A EP07817535A EP2035832A1 EP 2035832 A1 EP2035832 A1 EP 2035832A1 EP 07817535 A EP07817535 A EP 07817535A EP 07817535 A EP07817535 A EP 07817535A EP 2035832 A1 EP2035832 A1 EP 2035832A1
Authority
EP
European Patent Office
Prior art keywords
spectrin
fodrin
multiple sclerosis
alpha
antibodies
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07817535A
Other languages
German (de)
English (en)
Inventor
Torsten Matthias
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP2035832A1 publication Critical patent/EP2035832A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis

Definitions

  • the invention relates to a method for the diagnosis of multiple sclerosis, in particular for the diagnosis and follow-up of impending or incipient inflammatory attacks, as well as the use of a test kit therefor.
  • MS Multiple sclerosis
  • HLA-DR2 HLA-DR2
  • HLA-DW2 HLA-DW2
  • Multiple sclerosis is an autoimmune disease in which the body's own immune system attacks and destroys the nerve bundles of wrapping Schwann's sheath cells or myelin sheaths. This damages the transmission of nerve stimuli. This leads in the further course of the disease to the fact that both the sensory and the motor nerves can maintain their function less and less, so that the typical for multiple sclerosis sensitivity disorders, as well as spastic paresis to dysphagia and paralysis of Mus- arise. In many cases (about 80%), the disease is primarily relapsing-remitting, with the symptoms often partially receding after overcoming the thrust. Usually, however, the course is chronically progressive with a frequently fatal pathogenesis.
  • MS Multiple sclerosis
  • MS occurs in a variety of forms, depending on its course, eg. For example, they can be distinguished according to their demyelination pattern of the lesions in the central nervous system or lesions in the brain, but this is only possible by means of a biopsy, or can be distinguished according to their immunological mechanism, d.
  • a test that identifies multiple sclerosis at an early stage or the onset of a worsening of it and that can be used to take appropriate therapeutic measures is not yet available. There is also a need for a follow-up to control the effects of therapeutic interventions.
  • the invention therefore aims to provide such a method. This object is achieved by the method defined in the claims and the test kit used therein.
  • multiple sclerosis is accompanied by an increase in antibodies which are directed against spectrin. These antibodies can be detected in body fluids.
  • Spectrins are proteins that bind to F-actin and thus cause the gelation of actin. It is present in the cytoskeleton of the cell and stabilizes the cell shape. Spectrin itself is present as a heterotetrameric protein, which consists of a 260 kDa alpha spectrin and a 225 kDa beta spectrin (alpha 2 beta 2 ). A subform, also referred to as spectrin-like protein, is fodrin. Fodrin cross-links neighboring actin filaments with each other. In a preferred embodiment, antibodies to fodrin are determined according to the invention. Fodrin itself is heretofore known as a differentiation marker for intestinal neoplasms (M. Younes et al., Am J. Pathol, Vol. 135 (1989), 1197-1212.
  • antibodies are determined which are directed against alpha-fodrin, with those antibodies being particularly preferred which bind against a 100 to 140 kDa or 110 to 130 kDa, in particular an approximately 120 kDa neoantigen of alpha -Finrin are addressed.
  • a neoantigen is cleaved off, for example, in apoptosis by a protease, in particular a caspase.
  • Typical caspases cleave peptide bonds C-terminal of aspartate, which is why they are also referred to as C-Asp-ase.
  • the cleavage of the 120 kD fragment of alpha-fodrin occurs in the body by means of caspase-3.
  • autoantibodies against spectrins, especially fodrin can be found in all forms of MS, they are particularly characteristic of subtypes 3 and especially 2.
  • WO 01/14877 describes a test kit for the determination of autoantibodies to alpha-fodrin as a specific marker for the diagnosis of Sjogren's syndrome.
  • the invention therefore also relates to the use of such a test for diagnosis and follow-up of MS and for the determination of subtypes 3 (predominantly apoptosis) and in particular 2 (antibody-mediated).
  • IgA autoantibodies to alpha-fodrin is thus a typical demonstration of the presence of MS, in particular subtypes 3 and 2.
  • the sensitivity of the test by additional determination of IgA and IgG and optionally IgM against the aforementioned spectra, in particular alpha-fodrin can be increased. Only the additional determination of IgG autoantibodies increases the sensitivity by about 10 - 20%.
  • the method according to the invention can be carried out both for the qualitative and for the quantitative determination of the antibodies which occur.
  • a quantitative determination of the autoantibodies indicates the acute state of the disease or the severity of the course.
  • the antibody concentration which is above a limit is defined as a positive result. The determination of such limit values is carried out by appropriate calibration of the test system with sera from healthy and diseased patients.
  • the antibodies are determined in a body fluid.
  • Typical body fluids are blood, serum and plasma, with cerebrospinal fluid being preferred.
  • the determination of the IgA or IgG antibodies to alpha-fodrin, or the approximately 120 kDa neoantigen thereof, can by means of the skilled person known techniques, such as. B. an ELISA or RIA be performed.
  • biosensors such as amperometric sensors, potentiometric, ion-selective potentiometric or photometric sensors or also by means of semiconductor electrodes such as field effect transistors (FET), chemosensitive field effect transistors (CHEMFET), suspended gate Field effect transistors (SGFETs) or ion-sensitive field-effect transistors.
  • FET field effect transistors
  • CHEMFET chemosensitive field effect transistors
  • SGFETs suspended gate Field effect transistors
  • ISFET ion-sensitive field effect transistors
  • optical detectors include F. Aberl and H. Wolf in “Current Trends in Immunosensitivity ", Labor 2000, pp.
  • the alpha-fodrin antigen may be a native protein obtainable from human cell lines, or a recombinant antigen prepared in a heterologous host cell, for example, a bacterial cell such as E. coli or a eukaryotic host cell such as an insect cell by recombinant protein expression has been.
  • a recombinant alpha-fodrin antigen is used which contains the sequence of the native alpha-fodrin or parts thereof, in particular the N-terminal portion.
  • the recombinant antigen may also contain heterologous peptide or polypeptide domains, e.g. B. a poly-His sequence, which facilitates the purification after expression.
  • the IgA-specific receptor is generally in antibody capable of class A immunoglobulins in the presence of immunoglobulins of other classes, e.g. B. G and / or M, to recognize selectively.
  • polyclonal anti-IgA antisera may be used which are immunopurified sation of experimental animals, eg. As goats, rats, mice, rabbits, etc. are available with human IgA by known methods.
  • monoclonal anti-IgA antibodies can be used.
  • the specific test format is generally not critical.
  • a heterogeneous assay format is used, more preferably a heterogeneous assay format in which an immune complex consisting of spectrin, in particular alpha-fodrin antigen, IgA autoantibody to be detected and IgA-specific receptor is bound to a solid phase (sandwich assay format).
  • spectrin in particular alpha-fodrin antigen
  • IgA autoantibody to be detected and IgA-specific receptor is bound to a solid phase (sandwich assay format).
  • a com petitive test format can also be selected.
  • reaction vessels As solid phases, reaction vessels, microtiter plates, beads, biochips, etc. can be used.
  • the immobilization of the antigen or of the receptor on the solid phase can be effected by adsorptive interactions, covalent binding or mediated via a high-affinity binding pair (streptavidin / biotin, hapten / anti-hapten antibody).
  • the immobilized test reagent can be used in an already solid-phase form or immobilized only during the course of the test.
  • the method can be carried out as a liquid test (for example in a reaction vessel) or else as a dry test (for example on a test strip).
  • the labeled test reagent may itself carry a detectable or signaling group (direct labeling) or be bindable with a detectable group (indirect labeling).
  • the marker group can Any of the labeling groups known from the prior art for immunological detection methods can be selected as desired, for example from enzymes, metal or latex particles, as well as luminescent or fluorescent groups. Most preferably, the tagging group is selected from enzymes, e.g. As peroxidase, ß-galactosidase or alkaline phosphatase selected and led the process in ELISA format druch-.
  • Yet another object of the invention relates to the use of a test kit for the diagnosis of multiple sclerosis comprising
  • spectrin antigen in particular an alpha-fodrin antigen
  • test kit (c) may comprise a solid phase to which one of the test reagents (a) or (b) is bound or capable of binding.
  • test kit preferably comprises (d) a label group that is bound to or capable of binding to any of the test reagents (a) or (b).
  • test kit (s) may contain at least one further antibody class-specific test reagent if, in addition to IgA autoantibodies, it is also intended to determine spectrin, in particular alpha-fodrin autoantibodies of other immunoglobulin classes.
  • antibody class-specific test reagents are anti-IgG antibodies or protein G for the selective binding of IgG autoantibodies or anti-IgM antibodies for the selective binding of IgM autoantibodies.
  • the test kit may also contain other common reagents such as buffers, substrates and wash solutions.
  • Sera from 99 patients with clinically stable MS were studied and 27 patients with acute respiratory distress treated with high doses of intravenous steroids.
  • the serum was taken before and after the treatment. Both untreated patients were enrolled in the study and patients treated with intraferon-ß or glatiramer acetate.
  • the sera were frozen at minus 20 0 C, and later for the presence of IgG and IgA alpha-fodrin antibodies using a commercial ELISA (AESKULISA alpha-fodrin A of Aesku. Diagnostics, Wendelsheim, DE), as well as antinuclear antibodies ANA from Hep2 cells (Aesku slides of Aesku Diagnostics, Wendelsheim, DE).
  • the concentration of these antibodies was also determined and the sera were reported to be positive, exceeding the limit of 15 U / ml, for both IgG and IgA values.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Rheumatology (AREA)
  • Rehabilitation Therapy (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un procédé et un kit de test pour le diagnostic et le contrôle de la sclérose en plaques par détermination des auto-anticorps dans des liquides corporels, dans lesquels on détermine les anticorps qui se lient à une spectrine. Une spectrine atypique est l'alpha-fodrine.
EP07817535A 2006-09-15 2007-09-14 Procédé de diagnostic et de contrôle de l'évolution de la sclérose en plaques et utilisation d'un kit de test dans ce but Withdrawn EP2035832A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102006043369A DE102006043369A1 (de) 2006-09-15 2006-09-15 Verfahren zur Diagnose und Verlaufskontrolle von Multipler Sklerose sowie die Verwendung eines Test-Kits hierfür
PCT/DE2007/001664 WO2008031429A1 (fr) 2006-09-15 2007-09-14 Procédé de diagnostic et de contrôle de l'évolution de la sclérose en plaques et utilisation d'un kit de test dans ce but

Publications (1)

Publication Number Publication Date
EP2035832A1 true EP2035832A1 (fr) 2009-03-18

Family

ID=38896023

Family Applications (1)

Application Number Title Priority Date Filing Date
EP07817535A Withdrawn EP2035832A1 (fr) 2006-09-15 2007-09-14 Procédé de diagnostic et de contrôle de l'évolution de la sclérose en plaques et utilisation d'un kit de test dans ce but

Country Status (5)

Country Link
US (1) US20100003696A1 (fr)
EP (1) EP2035832A1 (fr)
JP (1) JP2010503835A (fr)
DE (1) DE102006043369A1 (fr)
WO (1) WO2008031429A1 (fr)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19939575C1 (de) * 1999-08-20 2001-08-02 Orgentec Diagnostika Gmbh Verfahren zur Diagnose von Sjögren-Syndrom

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2008031429A1 *

Also Published As

Publication number Publication date
DE102006043369A1 (de) 2008-03-27
JP2010503835A (ja) 2010-02-04
US20100003696A1 (en) 2010-01-07
WO2008031429A1 (fr) 2008-03-20

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