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WO2026003017A1 - Méthodes et kits pour la détection simultanée d'auto-anticorps dirigés contre les protéines de jonction dermo-épidermique dans des maladies de la peau bulleuse auto-immune - Google Patents

Méthodes et kits pour la détection simultanée d'auto-anticorps dirigés contre les protéines de jonction dermo-épidermique dans des maladies de la peau bulleuse auto-immune

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Publication number
WO2026003017A1
WO2026003017A1 PCT/EP2025/067791 EP2025067791W WO2026003017A1 WO 2026003017 A1 WO2026003017 A1 WO 2026003017A1 EP 2025067791 W EP2025067791 W EP 2025067791W WO 2026003017 A1 WO2026003017 A1 WO 2026003017A1
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WO
WIPO (PCT)
Prior art keywords
amino acid
particles
conjugated
acid residue
domain
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.)
Pending
Application number
PCT/EP2025/067791
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English (en)
Inventor
Marie-Laure GOLINSKI
Billal TEDBIRT
Maud MAHO-VAILLANT
Sébastien CALBO
Clément DEVALLEE
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.)
Universite de Rouen
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Rouen
Original Assignee
Universite de Rouen
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Rouen
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Filing date
Publication date
Application filed by Universite de Rouen, Institut National de la Sante et de la Recherche Medicale INSERM, Centre Hospitalier Universitaire de Rouen filed Critical Universite de Rouen
Publication of WO2026003017A1 publication Critical patent/WO2026003017A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6881—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from skin
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N2800/20—Dermatological disorders

Definitions

  • the blood sample is a serum sample or a plasma sample.
  • the term "particle” has its general meaning in the art and refers to a particle from 1 nm to 1000 nm, preferably from 100 to 500 nm and even more preferably from 350 to 450 nm in size. In some embodiments, the size of the particle is about 400 nm.
  • a particle may typically be spherical, though the shape is not limited to that of a sphere and may include other shapes like spheroid, irregular particles, cubes, irregular cubes, and disks. According to the present invention the term “particle” is interchangeable with the term “bead”.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology.48 (3): 443–53.).
  • the percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk).
  • EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5.
  • the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and/or biological modification.
  • a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.
  • the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion.
  • substitution means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted into the same position.
  • conservative mutations refers to amino acid modifications that do not significantly affect or alter the biologic function of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a protein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • Amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
  • Other families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • BP180 refers to a protein that is also known as collagen XVII alpha 1, a transmembrane collagen that is involved in the adhesion of the epidermis to the basement membrane.
  • BP180 is composed of an extracellular domain, a transmembrane domain and an intracellular domain.
  • the extracellular domain consists of 15 collagenous domains and 16 non- collagenous domains (NC), among which NC16A and NC14A are the most immunogenic and reactive with autoantibodies in patients with bullous pemphigoid, a common autoimmune bullous skin disease.
  • NC16A and NC14A are the most immunogenic and reactive with autoantibodies in patients with bullous pemphigoid, a common autoimmune bullous skin disease.
  • the C-terminal domain of BP180 is also recognized by autoantibodies in some patients with bullous pemphigoid and other autoimmune diseases.
  • the LAD-1 domain of BP180 is an anchoring filament protein which is a component of basement membranes and is also recognized by autoantibodies in some patients with bullous pemphigoid, Linear IgA bullous disease and other autoimmune diseases.
  • An exemplary amino acid sequence for BP180 is shown as SEQ ID NO:1 wherein the NC16A domain ranges from the amino acid residue at position 490 to the amino acid residue at position 566, the C-terminal domain ranges from the amino acid residue at position 1193 to the amino acid residue at position 1497 and the LAD-1 domain ranges from the amino acid residue at position 490 to the amino acid residue at position 1497.
  • SEQ ID NO:1 >sp
  • Integrin ⁇ 6 ⁇ 4 is also involved in signaling pathways that regulate cell survival, proliferation, and differentiation.
  • autoimmune bullous skin diseases such as bullous pemphigoid and epidermolysis bullosa acquisita
  • autoantibodies target integrin ⁇ 6 ⁇ 4 or other components of the basement membrane, leading to blistering and inflammation of the skin and mucous membranes.
  • An exemplary amino acid sequence of the alpha 6 subunit is shown as SEQ ID NO:2 and an exemplary amino acid sequence of beta 4 subunit is shown as SEQ ID NO:3.
  • Collagen VII is encoded by the COL7A1 gene and is composed of three identical alpha chains. Mutations in collagen VII can cause dystrophic epidermolysis bullosa, a genetic disorder that causes blisters and erosions on the skin and mucous membranes. Autoantibodies against collagen VII can also cause autoimmune bullous skin diseases, such as epidermolysis bullosa acquisita.
  • An exemplary amino acid sequence for COL7A1 is shown as SEQ ID NO:4 wherein the NC1 domain ranges from the amino acid residue at position 17 to the amino acid residue at position 1253 and the NC2 domain ranges from the amino acid residue at position 2785 to the amino acid residue at position 2944.
  • DST acts as an integrator of intermediate filaments, actin and microtubule cytoskeleton networks.
  • the DST gene produces several isoforms, including DST-a, DST-b, and DST-e, which are expressed in neural, muscle, and cutaneous tissues, respectively.
  • Pathogenic DST mutations cause hereditary sensory and autonomic neuropathy type 6 (HSAN-VI) and epidermolysis bullosa simplex (EBS).
  • HSAN-VI hereditary sensory and autonomic neuropathy type 6
  • EBS epidermolysis bullosa simplex
  • An exemplary amino acid sequence for the BP230 is shown as SEQ ID NO: 5 wherein the BP230 Extra Cellular domain ranges from the amino acid residue at position 2077 to the amino acid residue at position 2649.
  • Laminins are major constituents of the basement membrane, namely the basal lamina (the protein network foundation for most cells and organs).
  • Laminins are vital to biological activity, influencing cell differentiation, migration, and adhesion.
  • Laminins are heterotrimeric proteins with a high molecular mass ( ⁇ 400 to ⁇ 900 kDa) and possess three different chains ( ⁇ , ⁇ , and ⁇ ) encoded by five, four, and three paralogous genes in humans, respectively.
  • Laminins are integral to the structural scaffolding of almost every tissue of an organism—secreted and incorporated into cell- associated extracellular matrices.
  • An exemplary amino acid sequence for the laminin subunit beta 4 p200 is shown as SEQ ID NO: 6 wherein the Laminin subunit beta 4 p200 domain ranges from the amino acid residue at position 1154 to the amino acid residue at position 1761 and an exemplary amino acid sequence of gamma 1 p200 is shown as SEQ ID NO: 7 wherein the Laminin subunit gamma 1 p200 ranges from the amino acid residue at position 1 to the amino acid residue at position 1609.
  • the particle of the present invention is made of an organic polymer.
  • Organic polymers encompass, but are not limited to, polystyrene, poly(vinyl acetate), poly(methylstyrene), poly(acrylamide), poly(acrylonitrile), poly(vinyl chloride), poly(butyl acrylate), poly(acrylic acid), copolymers of styrene and C1-C4alkyl (meth)acrylate, copolymers of styrene and acrylamide, copolymers of styrene and acrylonitrile, copolymers of styrene and vinyl acetate, copolymers of acrylamide and C1-C4 alkyl (meth)acrylates, copolymers from acrylonitrile and C1-C4 alkyl (meth)acrylate, copolymers of acrylonitrile and acrylamide, terpolymers from styrene, acrylonitrile and acrylamide, poly(methyl methacrylate), poly(ethyl methacryl
  • Polymer particles can be crosslinked or not.
  • organic particles include, but are not limited to, nylon (for example marketed by ATOCHEM), polyethylene powders (for example marketed by PLAST LABOR), poly-2-alanine powders, polyfluorinated powders such as polytetrafluoroethylene (for example marketed by DUPONT DE NEMOURS), acrylic copolymer powders (for example marketed by DOW CHEMICA), polystyrene powders (for example marketed by PRESPERESE), polyester powders, expanded microspheres in thermoplastic material (for example marketed by EXPANCEL), microballs of silicon resins (for example marketed by TOSHIBA), synthetic hydrophilic polymer powders such as polyacrylates (for example marketed by MATSUMOTO), acrylic polyamides (for example marketed by ORIS), insoluble polyurethanes (for example marketed by TOSHNU), porous microspheres of cellulose, micro- or particles of PTFE (polytetrafluoro
  • the particles are selected to have a variety of properties useful for particular experimental formats. For example, particles can be selected that remain suspended in a solution of desired viscosity or to readily precipitate in a solution of desired viscosity.
  • the particles are magnetic and coded.
  • particles can be coded for identification purposes, such as by bar codes, luminescence, fluorescence and the like. A variety of coded particles are well known to those skilled in the art, and include for example, Luminex® and Cyvera® coded particles.
  • each particle can include a unique code
  • the coded particles contain a code other than that present in the detectable tag used to detect the presence or amount of modified substrate (e.g., support-bound product portion, free product portion, or modified support-bound substrate).
  • the code can be embedded (for example, within the interior of the particle) or otherwise attached to the particle in a manner that is stable through hybridization and analysis.
  • the code can be provided by any detectable means, such as by holographic encoding, by a fluorescence property, color, shape, size, light emission, quantum dot emission and the like to identify particle and thus the capture probes immobilized thereto.
  • the particles may be encoded using optical, chemical, physical, or electronic tags.
  • Examples of such coding technologies are optical bar codes fluorescent dyes, or other means.
  • One exemplary platform utilizes mixtures of fluorescent dyes impregnated into polymer particles as the means to identify each member of a particle set to which a specific capture probe has been immobilized.
  • Another exemplary platform uses holographic barcodes to identify cylindrical glass particles.
  • Chandler et al. U.S. Pat. No. 5,981,180
  • Soini U.S. Pat. No. 5,028,545
  • Fulwyler (U.S. Pat. No.4,499,052) describes an exemplary method for using particle distinguished by color and/or size.
  • U.S. Patent Publication Nos. 2004-0179267, 2004-0132205, 2004-0130786, 2004-0130761, 2004- 0126875, 2004-0125424, and 2004-0075907 describe exemplary particles encoded by holographic barcodes.
  • U.S. Pat. No. 6,916,661 describes polymeric particles (e.g., microparticles) that are associated with particles that have dyes that provide a code for the particles.
  • magnetic particle encompasses any particle having at least some magnetic characteristic, e.g., ferromagnetic, paramagnetic, and superparamagnetic property.
  • a magnetic particle can include magnetic materials such as iron, nickel, and cobalt, as well as metal oxides such as Fe 3 O 4 , BaFe 12 O 19 , Mn 2 O 3 , Cr 2 O 3 , CoO, NiO, and CoMnP.
  • the magnetic particle contains, or fully consists of, a polymeric magnetic material.
  • Polymeric magnetic material includes for example, material in which the magnetic material is mixed with polymeric material and magnetic material that is coated with polymeric material.
  • the magnetic material is only one component of the microparticle whose remainder consists of a polymeric material to which the magnetically responsive material is affixed (see coded particles below).
  • a first group of particles is conjugated to a polypeptide that derives from the NC16A domain of BP180.
  • the first group of particles is conjugated to a polypeptide having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 490 to the amino acid residue at position 566 in SEQ ID NO:1.
  • the first group of particles is conjugated to a polypeptide having the amino acid sequence that ranges from the amino acid residue at position 490 to the amino acid residue at position 566 in SEQ ID NO:1 and that comprises one or more mutations, preferably one or more conservatives, even preferably one or more conservatives substitutions.
  • a second group of particles is conjugated to polypeptide that derives from the C-terminal domain of BP180.
  • the second group of particles is conjugated to a polypeptide having at least 90% identity with the amino acid sequence that ranges from the amino acid residue at position 1193 to the amino acid residue at position 1497 in SEQ ID NO:1.
  • the second group of particles is conjugated to a polypeptide having the amino acid sequence that ranges from the amino acid residue at position 1193 to the amino acid residue at position 1497 in SEQ ID NO:1 and that comprises one or more mutations, preferably one or more conservative mutations, even more preferably one or more conservative substitutions.
  • a third group of particles is conjugated to a first polypeptide that derives from the alpha 6 subunit of the integrin ⁇ 6 ⁇ 4 and/or to a second polypeptide that derives from the beta 4 subunit of the integrin ⁇ 6 ⁇ 4.
  • the third group of particles is conjugated to a first polypeptide having an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 24 to the amino acid residue at position 878 in SEQ ID NO:2. In some embodiments, the third group of particles is conjugated to a first polypeptide having the amino acid sequence that ranges from the amino acid residue at position 24 to the amino acid residue at position 878 in SEQ ID NO:2 and that comprises one or more mutations, preferably one or more conservative mutations, even more preferably one or more conservative substitutions.
  • the third group of particles is conjugated to a second polypeptide having an amino acid sequence having at least 90% identity with the amino acid sequence that ranges from the amino acid residue at position 28 to the amino acid residue at position 710 in SEQ ID NO:3.
  • the third group of particles is conjugated to a second polypeptide having the amino acid sequence that ranges from the amino acid residue at position 28 to the amino acid residue at position 710 in SEQ ID NO:3 and that comprises one or more mutations, preferably one or more conservative mutations, even more preferably one or more conservative substitutions.
  • a fourth group of particles is conjugated to a polypeptide that derives from the NC1 domain of COL7A1.
  • the fourth group of particles is conjugated to a polypeptide having at least 90% identity with the amino acid sequence that ranges from the amino acid residue at position 17 to the amino acid residue at position 1253 in SEQ ID NO:4. In some embodiments, the fourth group of particles is conjugated to a polypeptide having the amino acid sequence that ranges from the amino acid residue at position 17 to the amino acid residue at position 1253 in SEQ ID NO:4 and that comprises one or more mutations, preferably one or more conservative mutations, even more preferably one or more conservative substitutions. In some embodiments, a fifth group of particles is conjugated to a polypeptide that derived from the NC2 domain of COL7A1.
  • the fifth group of particles is conjugated to a polypeptide having at least 90% identity with the amino acid sequence that ranges from the amino acid residue at position 2785 to the amino acid residue at position 2944 in SEQ ID NO:4. In some embodiments, the fifth group of particles is conjugated to a polypeptide having the amino acid sequence that ranges from the amino acid residue at position 2785 to the amino acid residue at position 2944 in SEQ ID NO:4 and that comprises one or more mutations, preferably one or more conservative mutations, even more preferably one or more conservative substitutions. In some embodiments, a sixth group of particles is conjugated to a polypeptide that derived from the LAD1 domain of BP180.
  • the sixth group of particles is conjugated to a polypeptide having at least 90% identity with the amino acid sequence that ranges from the amino acid residue at position 490 to the amino acid residue at position 1497 in SEQ ID NO:1. In some embodiments, the sixth group of particles is conjugated to a polypeptide having the amino acid sequence that ranges from the amino acid residue at position 490 to the amino acid residue at position 1497 in SEQ ID NO:1 and that comprises one or more mutations, preferably one or more conservative mutations, even more preferably one or more conservative substitutions. In some embodiments, a seventh group of particles is conjugated to a polypeptide that derived from the BP230.
  • the seventh group of particles is conjugated to a polypeptide having at least 90% identity with the amino acid sequence in SEQ ID NO:5. In some embodiments, the seventh group of particles is conjugated to a polypeptide having at least 90% identity with the amino acid sequence that ranges from the amino acid residue at position 2077 to the amino acid residue at position 2649 in SEQ ID NO:5. In some embodiments, the seventh group of particles is conjugated to a polypeptide having the amino acid sequence that ranges from the amino acid residue at position 2077 to the amino acid residue at position 2649 in SEQ ID NO:5 and that comprises one or more mutations, preferably one or more conservative mutations, even more preferably one or more conservative substitutions.
  • an eighth group of particles is conjugated to a polypeptide that derived from the ⁇ 4 chain of P200 laminin.
  • the seventh group of particles is conjugated to a polypeptide having at least 90% identity with the amino acid sequence in SEQ ID NO:6.
  • the eighth group of particles is conjugated to a polypeptide having at least 90% identity with the amino acid sequence that ranges from the amino acid residue at position 1154 to the amino acid residue at position 1761 in SEQ ID NO:6.
  • the eighth group of particles is conjugated to a polypeptide having the amino acid sequence that ranges from the amino acid residue at position 1154 to the amino acid residue at position 1761 in SEQ ID NO:6 and that comprises one or more mutations, preferably one or more conservative mutations, even more preferably one or more conservative substitutions.
  • a nineth group of particles is conjugated to a polypeptide that derived from the ⁇ 1 chain of P200 laminin.
  • the nineth group of particles is conjugated to a polypeptide having at least 90% identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 1609 in SEQ ID NO:7.
  • the nineth group of particles is conjugated to a polypeptide having the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 1609 in SEQ ID NO:7 and that comprises one or more mutations, preferably one or more conservative mutations, even more preferably one or more conservative substitutions.
  • the polypeptides that are conjugated to the particles can comprise a tag.
  • the polypeptides can comprise a His-tag, a FLAG-tag, a HA-tag, a c-Myc-tag, a GST-tag, an MBP-tag, a DDK-tag or any other suitable tag known in the art.
  • the tag can be located at the N-terminus, the C-terminus, or within the polypeptide sequence.
  • the tag can be removed after the conjugation or retained on the polypeptide-particle complex.
  • the tag can be used to bind the polypeptide to the particle through an intermediate molecule that recognizes the tag, such as an antibody, a streptavidin, a nickel-chelate, or any other suitable binding partner known in the art.
  • the intermediate molecule can be attached to the particle by covalent or non-covalent interactions.
  • the tag can be used to purify the polypeptide before or after the conjugation to the particle by using affinity chromatography or other methods well known in the art.
  • the tag can be used to detect the polypeptide on the particle by using immunoassays, fluorescence, or other methods well known in the art.
  • the polypeptides that are conjugated to the particles can be produced according to any well- known method in the art.
  • the polypeptides can be expressed in bacterial cells, such as Escherichia coli (E. coli), or mammalian cells, such as Chinese hamster ovary (CHO) cells, using recombinant DNA technology.
  • the polypeptides can be cloned into suitable expression vectors, such as plasmids or viruses, and transfected or infected into the host cells.
  • the polypeptides can be purified from the cell culture supernatant or lysate by using affinity chromatography, ion exchange chromatography, gel filtration chromatography, or any other suitable purification method known in the art.
  • the polypeptides can be verified by using electrophoresis, mass spectrometry, Western blotting, or any other suitable analytical method known in the art.
  • the polypeptides are conjugated directly to the particle, meaning that they form a covalent bond with a reactive group on the particle surface.
  • the polypeptides are conjugated indirectly to the particle, meaning that they are linked through an intermediate molecule or moiety that can bind both the polypeptide and the particle.
  • the intermediate molecule can be a biotin-avidin complex, a streptavidin-biotin complex, an antibody-antigen complex, a receptor-ligand complex, or any other suitable binding pair known in the art.
  • the intermediate molecule can be attached to the polypeptide and/or the particle by covalent or non-covalent interactions.
  • the conjugation is performed by any conventional method well known in the art, such as described in Hermanson, Greg T. Bioconjugate techniques. Academic press, 2013.
  • 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC)- N- hydroxysulfosuccinimide (Sulfo NHS) reactions are used for conjugating the polypeptides to the particles.
  • the particle is conjugated to an avidin moiety that can create an avidin-biotin complex with the biotinylated polypeptides and the particles.
  • Additional, appropriate cross-linking agents for use in the invention include a variety of agents that are capable of reacting with a functional group present on a surface of the particle. Reagents capable of such reactivity include homo- and hetero-bifunctional reagents, many of which are known in the art.
  • a typical bifunctional cross-linking agent is N-succinimidyl(4-iodoacetyl) aminobenzoate (SIAB).
  • SATA N- succinimidyl-S-acetyl-thioacetate
  • SPDP succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate
  • HYNIC 6- hydrazinonicotimide
  • cross-linking reagents see, e.g., S. S. Wong, “Chemistry of Protein Conjugation and Cross-Linking,” CRC Press (1991), and G. T. Hermanson, “Bioconjugate Techniques,” Academic Press (1995).
  • the particles belonging to the first, second, third, fourth, sixth, seventh, eighth and nineth group are directly conjugated to the polypeptides that derive from the NC16A domain of BP180, the C-terminal domain of BP180, the alpha6 integrin subunit, the beta4 integrin subunit, the NC1 domain of COL7A1, the NC2 domain of COL7A1, the LAD-1 domain of BP180, the BP230, the ⁇ 4 chain of P200 laminin and the ⁇ 1 chain of P200 laminin respectively, by covalent bonds between a reactive group on the particle surface and an amino acid residue on the polypeptide.
  • the polypeptide that derives from the NC2 domain of COL7A1 is indirectly conjugated to the particles belonging to the fifth group as described above.
  • the indirect conjugation is performed by using an antibody that recognizes a tag that is fused to the NC2 polypeptide, such as a His tag or any other suitable tag known in the art.
  • the antibody can bind both the NC2-tagged polypeptide and the particle, thereby creating a complex between them.
  • the antibody is a monoclonal antibody or a polyclonal antibody that specifically binds to the tag.
  • the antibody is conjugated to the particle by any conventional method well known in the art, such as described in Hermanson, Greg T. Bioconjugate techniques. Academic press, 2013.
  • the receptacle may be any solid container, for example a test tube, a microplate well or a reaction cuvette made of polypropylene.
  • the elimination of the unbound reagents may be carried out by any technique known to those skilled in the art, such as e.g. washing by means of repeated centrifugation steps.
  • immunocomplex refers to the complex formed between the specific autoantibodies of the subject and their specific antigen, i.e. the polypeptide that is conjugated to the particle.
  • the presence and amount of the immunocomplexes may be detected by methods known in the art, including label-based and label-free detection.
  • the method of the present invention includes use of a secondary antibody that is coupled to an indicator reagent comprising a signal generating compound.
  • the secondary antibody has specificity for a particular immunoglobulin.
  • the secondary antibody is an anti-human IgG antibody, including anti- IgG1, IgG2, IgG3 and IgG4 antibodies.
  • the secondary antibody is an anti-human IgA antibody, in particular an anti-IgA1 or IgA2 antibody.
  • the secondary antibody is an anti-human IgE antibody.
  • the antibody having specificity for a particular type immunoglobulin is a mouse, rabbit or goat antibody.
  • the antibody of the present invention is a monoclonal antibody or a polyclonal antibody.
  • Indicator reagents include chromogenic agents, catalysts such as enzyme conjugates, fluorescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums, ruthenium, and luminol, radioactive elements, direct visual labels, as well as cofactors, inhibitors and magnetic particles.
  • enzyme conjugates include alkaline phosphatase, horseradish peroxidase and beta-galactosidase.
  • the secondary antibody is conjugated to phycoerythrin.
  • Methods for detecting the particle identity codes are known in the art and are described below.
  • Examples of systems that read (detect or analyze) multiplex assay signals from Luminex beads include, e.g., the Luminex xMAP 100 and xMAP 200 instruments or the Bio-Plex 100 and Bio-Plex 200 from BioRad instruments.
  • a method for detecting and/or separating particle is the chemiluminescence.
  • Another method for detecting and/or separating particle sets based on ID codes is flow cytometry. Methods of and instrumentation for flow cytometry are known in the art, and those that are known can be used in the practice of the present invention.
  • Flow cytometry in general, involves the passage of a suspension of the particles as a stream past a light beam and electro-optical sensors, in such a manner that only one particle at a time passes through the region. As each particle passes this region, the light beam is perturbed by the presence of the particle, and the resulting scattered and fluorescent light are detected. The optical signals are used by the instrumentation to identify the subgroup to which each particle belongs, along with the presence and amount of label, so that individual assay results are achieved.
  • the detecting step thus involved the use of detector.
  • the term “detector” is intended to mean a device or apparatus that converts the energy of contacted photons into an electrical response.
  • the term can include an apparatus that produces an electric current in response to impinging photons such as in a photodiode or photomultiplier tube.
  • a detector can also accumulate charge in response to impinging photons and can include, for example, a charge coupled device.
  • the detector involves the use of a radiation source.
  • the term “radiation source” is intended to mean an origin or generator of propagated electromagnetic energy.
  • the term can include any illumination sources including, for example, those producing electromagnetic radiation in the ultraviolet, visible and/or infrared regions of the spectrum.
  • a radiation source can include, for example, a lamp such as an arc lamp or quartz halogen lamp, or a laser.
  • the term “laser” is intended to mean a source of radiation produced by light amplification by stimulated emission of radiation.
  • the term can include, for example, an ion laser such as argon ion or krypton ion laser, helium neon laser, helium cadmium laser, dye laser such as a rhodamine 6G laser, YAG laser or diode laser.
  • ion laser such as argon ion or krypton ion laser
  • helium neon laser such as a rhodamine 6G laser, YAG laser or diode laser.
  • dye laser such as a rhodamine 6G laser, YAG laser or diode laser.
  • the detector is a flow cytometer.
  • flow cytometer is intended to mean a device or apparatus having a means for aligning the particles in a sample stream and a detector aligned such that the particles individually enter a zone of detection.
  • a sample stream can include any mobile phase that passes particles in single file including, for example, a fluid stream or fluid jet.
  • the method of the present invention is particularly suitable for simultaneously detecting the presence or absence of autoantibodies directed against the NC16A domain of BP180, the C-terminal domain of BP180, the integrin ⁇ 6 ⁇ 4, the NC1 domain of collagen VII, the NC2 domain of collagen VII, the LAD-1 domain of BP180, the BP230, the ⁇ 4 chain of P200 laminin or the ⁇ 1 chain of P200 laminin in a single sample.
  • the groups of said particles differ from one another by their identity codes (e.g. fluorophores) as described above.
  • the method of the present invention thus involves the use of a multiplex technology.
  • the term “multiplex technology” is the collective term for a variety of techniques which can assess multiple immunoglobulin specificities simultaneously on small volumes of sample.
  • the advantage of multiplex technology is that it is able to provide very rapid test times and very high throughput of samples.
  • the method of the present invention involves an addressable laser bead immunoassay (ALBIA), which is commercially available on LuminexTM-based platforms.
  • ALBIA is a semi-quantitative homogenous fluorescence-based microparticle immunoassay that can be used for the simultaneous detection of several immunogobulins (e.g. up to 10 immunoglobulins).
  • Each antigen i.e.
  • NC16A domain of BP180, the C-terminal domain of BP180, the integrin ⁇ 6 ⁇ 4, the NC1 domain of collagen VII, the NC2 domain of collagen VII, the LAD-1 domain of BP180, the BP230, the ⁇ 4 chain of P200 laminin and the ⁇ 1 chain of P200 laminin) is covalently coupled to a set of distinct uniform size colour-coded particles.
  • the sample is then incubated with the particles in the single assay receptacle and thus are contacted with group of particles.
  • the particles are then washed and then incubated with secondary anti-human IgG or IgA or IgE conjugated to a fluorescent label (e.g. phycoerythrin).
  • the particles are analysed on a system in which separate lasers identified antigen by bead colour and quantified the antibody by measuring the fluorescence of the fluorescent label. Said quantification thus indicated the level of the detected autoantibodies.
  • the methods of the present invention are not limited to the detection of autoantibodies of the IgG isotype, but can also detect autoantibodies of any other isotype, such as IgA or IgE. This is advantageous because some patients with autoimmune blistering diseases may have autoantibodies of different isotypes, which may have different pathogenic roles and clinical implications. Therefore, the methods of the present invention provide a comprehensive and accurate assessment of the autoantibody profile of a patient.
  • the method of the present invention is particularly suitable for the diagnosis of autoimmune bullous skin diseases which are characterized by the presence of autoantibodies directed against structural proteins of the dermo-epidermal junction (DEJ). These autoantibodies disrupt the integrity of the skin and mucous membranes, leading to blisters and erosions. The detection and identification of these autoantibodies is crucial for the diagnosis, classification, prognosis and treatment of these diseases.
  • the method of the present invention allows for a rapid and reliable detection of five major autoantibody specificities in a single assay, using a small volume of sample and a multiplex technology. This provides a significant advantage over the conventional methods, which require multiple assays, larger volumes of sample, and more time and resources.
  • the method of the present invention can thus facilitate the diagnosis of autoimmune bullous skin diseases and improve the management of patients with these conditions.
  • the method of diagnosis described herein is applied to a subject who presents symptoms of an autoimmune bullous skin disease without having undergone the routine screening to rule out all possible causes for said autoimmune bullous skin disease.
  • the methods described herein can be part of the routine set of tests performed on a subject who presents symptoms of an autoimmune bullous skin disease such as painful blisters that start in the mouth or skin areas, skin blisters near the surface of the skin that come and go, as well as oozing, crusting, or peeling at the blister site.
  • the method of the present invention can be carried out in addition of other diagnostic tools such as histology.
  • the method of the present invention is also particularly suitable for determining whether a subject suffering from an autoimmune bullous skin disease achieves a response with a treatment.
  • the method is thus particularly suitable for discriminating responder from non-responder.
  • responder in the context of the present disclosure refers to a subject that will achieve a response, i.e. a subject who is under remission and more particularly a subject who does not suffers from blisters.
  • non-responder refers to a subject for whom the disease does not show reduction or improvement after the treatment (e.g. the blisters remains stable or increases).
  • detecting the absence of the autoantibodies indicates that the patient achieve a response with the treatment.
  • the method of the present invention is also particularly suitable for determining whether a subject is at risk of relapse after a treatment.
  • the term "risk” in the context of the present invention relates to the probability that an event will occur over a specific time period and can mean a subject's "absolute” risk or "relative” risk.
  • Absolute risk can be measured with reference to either actual observation post- measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion.
  • "Risk evaluation” or “evaluation of risk” in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population.
  • the methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk of conversion.
  • the term "relapse” refers to the return of signs and symptoms of a disease after a subject has enjoyed a remission after a treatment.
  • the target disease is alleviated or healed, or progression of the disease was halted or slowed down, and subsequently the disease or one or more characteristics of the disease resume (e.g. blisters)
  • the subject is referred to as being "relapsed.”
  • the treatment is an immunosuppressive treatment.
  • the treatment consists in any method or drug that could be suitable for the treatment of an autoimmune bullous skin disease.
  • the treatment consists in an antibody depleting strategy, which typically include plasma exchange, plasmapheresis or immunoadsorption.
  • the treatment consists in administering immunoglobulins (e.g. by intravenous route).
  • the treatment is an immunosuppressive treatment.
  • immunosuppressive treatment refers to any substance capable of producing an immunosuppressive effect, e.g., the prevention or diminution of the immune response and in particular the prevention or diminution of the production of Ig.
  • Immunosuppressive drugs include, without limitation Prednisone, Dexamethasone, Rituximab, Mycophenolate mofetil, Azathioprine, and in more refractory cases, Cyclophosphamide, and Methotrexate.
  • the immunosuppressive drug is a corticosteroid.
  • corticosteroid has its general meaning in the art and refers to class of active ingredients having a hydrogenated cyclopentoperhydrophenanthrene ring system endowed with an anti-inflammatory activity.
  • Corticosteroid drugs typically include cortisone, cortisol, hydrocortisone (11 ⁇ ,17-dihydroxy, 21-(phosphonooxy)-pregn-4-ene, 3,20-dione disodium), dihydroxycortisone, dexamethasone (21-(acetyloxy)-9-fluoro-1 ⁇ ,17-dihydroxy-16 ⁇ -m- ethylpregna-1,4-diene-3,20-dione), and highly derivatized steroid drugs such as beconase (beclomethasone dipropionate, which is 9-chloro-11- ⁇ , 17,21, trihydroxy-16 ⁇ -methylpregna- 1,4 diene-3,20-dione 17,21-dipropionate).
  • beconase beclomethasone dipropionate, which is 9-chloro-11- ⁇ , 17,21, trihydroxy-16 ⁇ -methylpregna- 1,4 diene-3,20-dione 17,21-dipropionate
  • corticosteroids include flunisolide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazacort and betamethasone.
  • corticosteroids for example, cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, betamethesone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, triamcinolone acetonide, betamethasone, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone.
  • the treatment consists of administering a B cell depleting agent.
  • B cell depleting agent refers to any agent that is capable of triggering lymphodepletion of B cells.
  • the B cell depleting agent is an antibody having specificity for CD20. Examples of antibodies having specificity for CD20 include: “C2B8” which is now called “Rituximab” (U.S. Pat. No.
  • a murine IgG1 kappa mAb covalently linked to MX-DTPA for chelating to yttrium-[90] murine IgG2a “BI,” also called “Tositumomab,” optionally labeled with radioactive 131I to generate the “1311-B1” antibody (iodine 131 tositumomab, BEXXARTM) (U.S. Pat. No. 5,595,721, expressly incorporated herein by reference); murine monoclonal antibody “1F5” (Press et al.
  • suitable antibodies include e.g. antibody GA101 (obinutuzumab), a third generation humanized anti-CD20-antibody of Biogen Idec/Genentech/Roche.
  • BLX- 301 of Biolex Therapeutics a humanized anti CD20 with optimized glycosylation or Veltuzumab (hA20), a 2nd-generation humanized antibody specific for CD20 of Immunomedics or DXL625, derivatives of veltuzumab, such as the bispecific hexavalent antibodies of IBC Pharmaceuticals (Immunomedics) which are comprised of a divalent anti- CD20 IgG of veltuzumab and a pair of stabilized dimers of Fab derived from milatuzumab, an anti-CD20 mAb enhanced with InNexus' Dynamic Cross Linking technology, of Inexus Biotechnology both are humanized anti-CD20 antibodies are suitable.
  • BM-ca a humanized antibody specific for CD20 (Int J. Oncol.2011 February; 38(2):335-44)), C2H7 (a chimeric antibody specific for CD20 (Mol Immunol. 2008 May; 45(10):2861-8)), PRO131921 (a third generation antibody specific for CD20 developed by Genentech), Reditux (a biosimilar version of rituximab developed by Dr Reddy's), PBO-326 (a biosimilar version of rituximab developed by Probiomed), a biosimilar version of rituximab developed by Zenotech, TL-011 (a biosimilar version of rituximab developed by Teva), CMAB304 (a biosimilar version of rituximab developed by Shanghai CP Guojian), GP-2013 (a biosimilar version of rituximab developed by Sandoz (Novartis)), SAIT-101 (a biosimilar version of rituximab developed by Samsung BioLogics
  • Kits of the present invention A further object of the present invention relates to a kit for performing the method of the present invention.
  • the present invention relates to a kit for the simultaneous detection of autoantibodies directed against nine dermo-epidermal junction (DEJ) proteins in a sample of a subject suspected of having an autoimmune bullous skin disease, comprising: a plurality of particles, each particle being conjugated with a different DEJ polypeptide selected from the group consisting of NC16A domain of BP180, C-terminal domain of BP180, LAD-1 domain of BP180, integrin ⁇ 6 ⁇ 4, NC1 domain of collagen VII, NC2 domain of collagen VII, BP230, ⁇ 1 chain of P200 laminin and ⁇ 4 chain of P200 laminin; and means for detecting the binding of autoantibodies to the particles.
  • DEJ dermo-epidermal junction
  • the present invention also relates to a kit for the detection of autoantibodies directed against one or more dermo-epidermal junction (DEJ) proteins in a sample of a subject suspected of having an autoimmune bullous skin disease, comprising: one or more particles, each particle being conjugated with a different DEJ polypeptide selected from the group consisting of NC16A domain of BP180, C-terminal domain of BP180, LAD-1 domain of BP180, integrin ⁇ 6 ⁇ 4, NC1 domain of collagen VII, NC2 domain of collagen VII, BP230, ⁇ 1 chain of P200 laminin and/or ⁇ 4 chain of P200 laminin; and means for detecting the binding of autoantibodies to the particles.
  • DEJ dermo-epidermal junction
  • the kit comprises one or more plurality of particles as above described and means for determining the immunocomplexes.
  • Reagents for particular types of assays can also be provided in kits of the invention.
  • the kits can include different groups of particles each identified by a specific identity, plates that comprises the single assay receptacles (e.g. a multiwell plate), and secondary antibodies as described above.
  • the kits comprise a device such as a detector as described above. The groups of particles, the plate, and the devices are useful for performing the immunoassay of the present invention.
  • kits can include various diluents and buffers, labelled conjugates or other agents for the detection of the specifically immunocomplexes, and other signal-generating reagents, such as enzyme substrates, cofactors and chromogens.
  • Other components of a kit can easily be determined by one of skill in the art.
  • the invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
  • FIGURES Figure 1: Validation of coupled beads with commercial antibodies directed against each human recombinant proteins or against the Tag (Histidine or DDK). NC16A coupled beads were validated with anti-NC16A (A) or anti-His Tag antibodies (B).
  • BP180 C-terminal domain coupled beads were validated with anti-BP180 (C) or anti-His Tag antibodies (D).
  • BP180 LAD-1 domain coupled beads were validated with anti-BP180 (E) or anti-His Tag antibodies (F).
  • BP230 coupled beads were validated with anti-BP230 (G) or anti-His Tag antibodies (H).
  • ⁇ 6 ⁇ 4 Integrin coupled beads were validated with anti- ⁇ 6 (I) or anti- ⁇ 4 antibodies (J).
  • the ⁇ 1 chain of P200 laminin coupled beads were validated with anti- ⁇ 1 (K) or anti-DDK Tag antibodies (L).
  • the ⁇ 4 chain of P200 laminin coupled beads were validated with anti- ⁇ 4 (M) or anti-His Tag antibodies (N).
  • Collagen VII NC1 domain coupled beads were validated with anti-NC1 (O) or anti-His Tag antibodies (P).
  • the NC2 domain of collagen VII was indirectly coupled to the beads, which were previously coupled to anti-His Tag antibodies.
  • the beads coupled to anti-His Tag antibodies were validated with anti-mouse IgG antibody (Q).
  • the secondary coating of anti-His Tag beads with the NC2 domain of collagen VII was validated with anti-NC2 antibody (R).
  • the quantity of NC2 (ng/1000 beads) required to saturate the beads was determined by incubating the beads with an increasing quantity of NC2, as revealed by an anti-NC2 antibody (S).
  • Figure 2 A-D.
  • FIG. 4 Validation of IgG ALBIA multiplex. For each coupled beads, 4 healthy donors sera and 11 patients sera were analyzed in nine independent ALBIA or in mutliplex. The correlation coefficient R2 was greater than 0.82 for each coupled beads.
  • Figure 5 Validation of IgA ALBIA multiplex. For each coupled beads, 4 healthy donors sera and 11 patients sera were analyzed in nine independent ALBIA or in mutliplex. The correlation coefficient R2 was greater than 0.79 for each coupled beads.
  • Figure 6 Analysis of patients' sera using ALBIA to detect IgG directed against nine proteins of the dermal-epidermal junction.
  • Table 1 characteristics of these nine human recombinant proteins Protein AA sequence Tag Molecular Weight Source including tag BP180 NC16A AA 490-566 His 10 E. coli BP180 C-terminal AA 1193-1497 His 31 Mammal cells Collagen VII NC1 AA 17-1253 His 134 Mammal cells Collagen VII NC2 AA 2785-2944 His 18 Mammal cells I ntegrin ⁇ 6 ⁇ 4 ⁇ 6: AA 24-878 His 115 Mammal cells ⁇ 4: AA 28-710 76 BP180 LAD-1 AA 489-1497 His 113 Eukaryotic cells P200 laminin - ⁇ 1 AA 1-1609 DDK 178 Mammal cells P200 laminin – ⁇ 4 AA 1154-1761 His 70 Mammal cells BP230 AA 2077-2649 His 92 Insect cells Purity and integrity of proteins were controlled on SDS-PAGE gels.
  • human recombinant proteins were loaded and resolved on 12% SDS-PAGE gels. After electrophoretic separation at 100V for 1 hour, gels were stained with commassie blue. The bands intensity were measured with an Odyssey infrared imaging system (LI-COR Inc.). Mutliplex Adressable laser bead immunoassay (ALBIA) for the simultaneously quantification of auto-antibodies directed against the dermo-epidermal junction proteins in autoimmune bullous skin diseases. An ALBIA was developed for each of these nine proteins.
  • ALBIA Mutliplex Adressable laser bead immunoassay
  • NC16A C-terminal and LAD-1 domains of BP180, BP230, ⁇ 6 ⁇ 4 Integrin, NC1 domain of collagen VII and ⁇ 1 and ⁇ 4 chains of P200 laminin to fluorescent beads (Biorad) according to the manufacturer’s protocol.
  • the NC2 domain of collagen VII was indirectly coupled to the beads, which were previously coupled to anti-His Tag antibodies.
  • the different coupled beads were validated by ALBIA; the coupled beads were incubated with commercial antibodies directed against each human recombinant protein or against Histidine (His) Tag or DDK Tag, which present on the recombinant proteins ( Figures 1A to 1S).
  • beads were incubated for 45min in the same conditions with 100 ⁇ l of specific biotinylated mouse anti-human secondary antibodies (SoutherBiotech) at the following dilution: anti-IgG at 1:2000, anti-IgA at 1:200, and washed. Finally, beads were incubated for 15 min with 50 ⁇ L of streptavidin-R-phycoerythrin (Biorad) diluted at 1:400, and 50 ⁇ l of DPBS (without Ca 2+ /Mg 2+ ) were added. MFI was determined on a Bio-Plex apparatus using Manager software version 4.0 (Bio-Rad). Negative control (no serum, secondary antibody only) and positive controls (highly positive sera) were included in every assay.
  • Biorad streptavidin-R-phycoerythrin
  • the ALBIA multiplex was validated with 4 healthy donors sera and 11 patients sera which were incubated i) with the nine coupled beads separated or ii) with the combination of these nine coupled beads.
  • the mean fluorescence intensities (MFI) obtained for each serum were highly correlated into the two techniques for the IgG ( Figure 4) and IgA ( Figure 5) detection.
  • Auto-antibody levels (in arbitrary units (AU)) were determined using the following formula: (MFI serum /MFI positivity threshold ) x 100, in which the positivity thresholds are the mean of the MFI obtained from 100 healthy donor sera + 2 standard deviations, theses threshold were determined for each of the nine types of coupled beads (with the nine proteins from the dermo-epidermal junction).
  • ALBIA detected IgG reactivity against BP180, BP230, and/or ⁇ 4 P200 in 20 out of 23 (87%) seronegative PB patients (Data not shown).
  • IgG and/or IgA targeting at least one of the nine DEJ proteins were identified in 21 out of 23 (91%) seronegative PB patients (Data not shown).
  • the IgG/IgA multiplex test identified anti-collagen VII autoantibodies (directed against the NC1 and/or NC2 domains) in 55% of seronegative EBA patients for whom the diagnosis was unclear (Data not shown).

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Abstract

Les inventeurs ont développé un test multiplex pour la détection d'auto-anticorps dirigés contre neuf protéines DEJ (les domaines NC16A, C-terminal et LAD -1 de BP180, BP230, α6β4 Intégrine, les domaines NC1 et NC2 de collagène VII et les chaînes γ1 et β4 de la laminine P200) dans les sérums d'un patient, afin d'établir un diagnostic spécifique. Le test multiplex est basé sur l'utilisation de microbilles revêtues de protéines DEJ recombinantes ou purifiées, qui peuvent être analysées par cytométrie en flux ou la technologie Luminex ®. Le test multiplex permet la mesure simultanée de multiples auto-anticorps dans un seul échantillon, réduisant le coût, le temps et le volume de sérum requis. Le test multiplex présente également le potentiel d'améliorer la sensibilité et la spécificité du diagnostic, ainsi que de fournir des informations pronostiques et thérapeutiques pour la gestion de patients atteints de maladies bulleuses auto-immunes.
PCT/EP2025/067791 2024-06-25 2025-06-24 Méthodes et kits pour la détection simultanée d'auto-anticorps dirigés contre les protéines de jonction dermo-épidermique dans des maladies de la peau bulleuse auto-immune Pending WO2026003017A1 (fr)

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