Methods and kits for the simultaneous detection of auto-antibodies directed against the dermo-epidermal junction proteins in autoimmune bullous skin diseases

Abstract

The inventors developed a multiplex test for the detection of autoantibodies directed against nine DEJ proteins (NC16A, C-terminal and LAD-1 domains of BP180, BP230, α6β4 Integrin, NC1 and NC2 domains of collagen VII and γ1 and β4 chains of P200 laminin).) in patient's sera, in order to establish a specific diagnosis. The multiplex test is based on the use of microbeads coated with recombinant or purified DEJ proteins, which can be analyzed by flow cytometry or Luminex® technology. The multiplex test allows the simultaneous measurement of multiple autoantibodies in a single sample, reducing the cost, time, and volume of serum required. The multiplex test also has the potential to improve the sensitivity and specificity of the diagnosis, as well as to provide prognostic and therapeutic information for the management of patients with autoimmune bullous diseases.

Description

[0001] METHODS AND KITS FOR THE SIMULTANEOUS DETECTION OF AUTO- ANTIBODIES DIRECTED AGAINST THE DERMO-EPIDERMAL JUNCTION PROTEINS IN AUTOIMMUNE BULLOUS SKIN DISEASES FIELD OF THE INVENTION: The present invention is in the field of medicine, in particular immunology and dermatology. BACKGROUND OF THE INVENTION: Autoimmune bullous skin diseases are a group of rare autoimmune disorders that cause blistering of the skin and mucous membranes, including mouth, nose, throat, eyes, and genitals. These blisters can be painful, itchy, and prone to infection, affecting the quality of life and well- being of patients. The blisters can also lead to scarring, vision loss, or other complications, depending on the location and severity of the lesions. The exact causes of these diseases are not fully understood, but they involve an aberrant immune response against some components of the skin or mucosal tissues. Some of these diseases are characterized by pathogenic autoantibodies targeting dermo- epidermal junction (DEJ) proteins. The DEJ is a complex structure that connects the epidermis and the dermis, providing mechanical stability and resistance to the skin. The DEJ proteins are essential for maintaining the integrity and function of the skin barrier, which protects the body from external agents and prevents water loss. The autoantibodies bind to the DEJ proteins and disrupt their normal function, resulting in the separation of the epidermis and the dermis and the formation of blisters. These autoimmune diseases can be difficult to differentiate, especially in patients with exclusive or predominant mucosal disorders. This is the case for Bullous pemphigoid (BP), mucous membrane pemphigoid (MMP), epidermolysis bullosa acquisita or linear IgA bullous dermatosis. BP is the most common autoimmune bullous disease, affecting mainly elderly people. It is characterized by tense blisters on the skin, often accompanied by itching and erythema. MMP is a heterogeneous group of diseases that affect the mucous membranes, especially the oral cavity, conjunctiva, and genitalia. MMP can cause severe scarring and functional impairment of the affected organs. Epidermolysis bullosa acquisita is a rare disease that mimics the clinical features of the inherited forms of epidermolysis bullosa, a group of genetic disorders that cause skin fragility and blistering. Linear IgA bullous dermatosis is another rare disease that can affect both children and adults. It presents with annular or polycyclic blisters on the skin and mucous membranes, sometimes associated with drug exposure or inflammatory bowel disease. Diagnosis of these diseases depends on various immunological tests that detect the presence and specificity of autoantibodies in the patient’s sera or tissue samples. These tests include direct or indirect immunofluorescence, immunoblot, biochip or Enzyme Link ImmunoSorbent Assay (ELISA). However, some of these tests have limited sensitivity, specificity, or availability, making the diagnosis challenging in some cases. Moreover, some patients may have overlapping or atypical clinical and immunological features, requiring additional tests or follow-up to establish a definitive diagnosis. Therefore, there is a need for easy-to-use and more sensitive tests that can accurately identify the autoantibody profile of patients with autoimmune bullous diseases, especially those with exclusive or predominant mucosal involvement. Such tests would facilitate the diagnosis and classification of these diseases, as well as the monitoring and treatment of the patients. SUMMARY OF THE INVENTION: The present invention is defined by the claims. In particular, the present invention relates to methods and kits for identifying the autoantibody profile of patients with autoimmune bullous skin diseases and allows to determine a specific diagnosis for these patients, who were for 60% of cases, with an undetermined diagnosis. DETAILED DESCRIPTION OF THE INVENTION: Main definitions: As used herein, the term "autoimmune bullous skin diseases" refers to a group of chronic autoimmune disorders characterized by the presence of autoantibodies against structural proteins of the skin and mucosa (notably in the epidermis or dermo-epidermal junction (DEJ)), resulting in the formation of blisters and erosions on the skin and / or mucous membranes. Examples of autoimmune bullous skin diseases include but are not limited to pemphigus, pemphigoid, epidermolysis bullosa acquisita, dermatitis herpetiformis, and linear IgA bullous dermatosis. As used herein, the term “antibody”, "immunoglobulin" or “Ig” has its general meaning in the art and relates to proteins of the immunoglobulin superfamily. The immunoglobulins are characterized by a structural domain, i.e., the immunoglobulin domain, having a characteristic immunoglobulin (Ig) fold. The term encompasses secretory immunoglobulins. Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains which are linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains, including the VL domain (light chain variable domain), the CL domain (light chain constant domain), the VH domain (heavy chain variable domain) and the CH domains (heavy chain constant domains) CH1, optionally a hinge region, CH2, CH3, and optionally CH4. There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: mu (μ) for IgM, delta (δ) for IgD, gamma (γ) for IgG, alpha (a) for IgA and epsilon (ε) for IgE. In the context of the invention, the immunoglobulin may be an IgM, IgD, IgG, IgA or IgE. Preferably, the immunoglobulin is an IgG. As well-known from the skilled person, the IgG isotype encompasses four subclasses: the subclasses lgG1, lgG2, lgG3 and lgG4. The IgA isotype encompasses 2 subclasses: IgA1 and IgA2 immunoglobulins. As used herein, the term "autoantibody" refers to an antibody produced by a subject, where the antibody is directed against one or more 'self antigens (e.g., antigens that are native to the individual, e.g., an antigen on a cell or tissue, or an endogenous peptide or protein). As used herein, the term "antigen" refers to a substance that can cause the immune system to produce an antibody response against it, and possibly can trigger a biological reaction when an antibody binds to it under the appropriate in vivo conditions. The term antigen as used herein shall refer to a whole target molecule or a fragment of such molecule recognized by an antigen binding site. Specifically, substructures of an antigen, e.g. a polypeptide, generally referred to as "epitopes", which are immunologically relevant, may be recognized by an antibody. Thus, in some embodiments, the antigen of the present invention comprises at least one epitope. Methods for identifying and characterizing epitopes are well known in the art. Typically, said methods include but are not limited to epitope prediction algorithms and MHC associated peptidome identified by mass spectrometry (MS). As used herein, the term “sample" refer to a biological sample obtained for the purpose of in vitro evaluation. Typical biological samples to be used in the method according to the invention are blood samples (e.g. whole blood sample or serum sample). As used herein, the term “blood sample” means any blood sample derived from the subject. Collections of blood samples can be performed by methods well known to those skilled in the art. In some embodiments, the blood sample is a serum sample or a plasma sample. As used herein, 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”. As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. Polypeptides when discussed in the context of the present invention refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function and / or conformation of the intact protein. As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. 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). For example, 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. In general, 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. According to the invention 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. As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. The term "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. As used herein, the term "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. For example, 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. Other groups of 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. These families include 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) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). As used herein, the term "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. 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|Q9UMD9|COHA1_HUMAN Collagen alpha-1(XVII) chain OS=Homo sapiens OX=9606 GN=COL17A1 PE=1 SV=3 MDVTKKNKRDGTEVTERIVTETVTTRLTSLPPKGGTSNGYAKTASLGGGSRLEKQSLTHGSSGYINSTG STRGHASTSSYRRAHSPASTLPNSPGSTFERKTHVTRHAYEGSSSGNSSPEYPRKEFASSSTRGRSQTR ESEIRVRLQSASPSTRWTELDDVKRLLKGSRSASVSPTRNSSNTLPIPKKGTVETKIVTASSQSVSGTY DATILDANLPSHVWSSTLPAGSSMGTYHNNMTTQSSSLLNTNAYSAGSVFGVPNNMASCSPTLHPGLST SSSVFGMQNNLAPSLTTLSHGTTTTSTAYGVKKNMPQSPAAVNTGVSTSAACTTSVQSDDLLHKDCKFL ILEKDNTPAKKEMELLIMTKDSGKVFTASPASIAATSFSEDTLKKEKQAAYNADSGLKAEANGDLKTVS TKGKTTTADIHSYGSSGGGGSGGGGGVGGAGGGPWGPAPAWCPCGSCCSWWKWLLGLLLTWLLLLGLLF GLIALAEEVRKLKARVDELERIRRSILPYGDSMDRIEKDRLQGMAPAAGADLDKIGLHSDSQEELWMFV RKKLMMEQENGNLRGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMG QRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGE VGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPRGEQGL TGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKIVTSEGSSMLTVPGPPGPPGA MGPPGPPGAPGPAGPAGLPGHQEVLNLQGPPGPPGPRGPPGPSIPGPPGPRGPPGEGLPGPPGPPGSFL SNSETFLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKG DPGVPGALGIPSGPSEGGSSSTMYVSGPPGPPGPPGPPGSISSSGQEIQQYISEYMQSDSIRSYLSGVQ GPPGPPGPPGPVTTITGETFDYSELASHVVSYLRTSGYGVSLFSSSISSEDILAVLQRDDVRQYLRQYL MGPRGPPGPPGASGDGSLLSLDYAELSSRILSYMSSSGISIGLPGPPGPPGLPGTSYEELLSLLRGSEF RGIVGPPGPPGPPGIPGNVWSSISVEDLSSYLHTAGLSFIPGPPGPPGPPGPRGPPGVSGALATYAAEN SDSFRSELISYLTSPDVRSFIVGPPGPPGPQGPPGDSRLLSTDASHSRGSSSSSHSSSVRRGSSYSSSM STGGGGAGSLGAGGAFGEAAGDRGPYGTDIGPGGGYGAAAEGGMYAGNGGLLGADFAGDLDYNELAVRV SESMQRQGLLQGMAYTVQGPPGQPGPQGPPGISKVFSAYSNVTADLMDFFQTYGAIQGPPGQKGEMGTP GPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQVYAGRRRRRSIAVKP As used herein, the term “integrin α6β4” has its general meaning in the art and refers to a protein that consists of two subunits, alpha 6 and beta 4, and plays a role in cell adhesion and migration. It is mainly expressed on the surface of epithelial cells, where it interacts with the basement membrane and forms structures called hemidesmosomes. Integrin α6β4 is also involved in signaling pathways that regulate cell survival, proliferation, and differentiation. In autoimmune bullous skin diseases, such as bullous pemphigoid and epidermolysis bullosa acquisita, autoantibodies target integrin α6β4 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. MAAAGQLCLLYLSAGLLSRLGAAFNLDTREDNVIRKYGDPGSLFGFSLAMHWQLQPEDKRLLLVGAPRA EALPLQRANRTGGLYSCDITARGPCTRIEFDNDADPTSESKEDQWMGVTVQSQGPGGKVVTCAHRYEKR QHVNTKQESRDIFGRCYVLSQNLRIEDDMDGGDWSFCDGRLRGHEKFGSCQQGVAATFTKDFHYIVFGA PGTYNWKGIVRVEQKNNTFFDMNIFEDGPYEVGGETEHDESLVPVPANSYLGLLFLTSVSYTDPDQFVY KTRPPREQPDTFPDVMMNSYLGFSLDSGKGIVSKDEITFVSGAPRANHSGAVVLLKRDMKSAHLLPEHI FDGEGLASSFGYDVAVVDLNKDGWQDIVIGAPQYFDRDGEVGGAVYVYMNQQGRWNNVKPIRLNGTKDS MFGIAVKNIGDINQDGYPDIAVGAPYDDLGKVFIYHGSANGINTKPTQVLKGISPYFGYSIAGNMDLDR NSYPDVAVGSLSDSVTIFRSRPVINIQKTITVTPNRIDLRQKTACGAPSGICLQVKSCFEYTANPAGYN PSISIVGTLEAEKERRKSGLSSRVQFRNQGSEPKYTQELTLKRQKQKVCMEETLWLQDNIRDKLRPIPI TASVEIQEPSSRRRVNSLPEVLPILNSDEPKTAHIDVHFLKEGCGDDNVCNSNLKLEYKFCTREGNQDK FSYLPIQKGVPELVLKDQKDIALEITVTNSPSNPRNPTKDGDDAHEAKLIATFPDTLTYSAYRELRAFP EKQLSCVANQNGSQADCELGNPFKRNSNVTFYLVLSTTEVTFDTPDLDINLKLETTSNQDNLAPITAKA KVVIELLLSVSGVAKPSQVYFGGTVVGEQAMKSEDEVGSLIEYEFRVINLGKPLTNLGTATLNIQWPKE ISNGKWLLYLVKVESKGLEKVTCEPQKEINSLNLTESHNSRKKREITEKQIDDNRKFSLFAERKYQTLN CSVNVNCVNIRCPLRGLDSKASLILRSRLWNSTFLEEYSKLNYLDILMRAFIDVTAAAENIRLPNAGTQ VRVTVFPSKTVAQYSGVPWWIILVAILAGILMLALLVFILWKCGFFKRSRYDDSVPRYHAVRIRKEERE IKDEKYIDNLEKKQWITKWNENESYS OX=9606 GN=ITGB4 PE=1 SV=5 MAGPRPSPWARLLLAALISVSLSGTLANRCKKAPVKSCTECVRVDKDCAYCTDEMFRDRRCNTQAELLA AGCQRESIVVMESSFQITEETQIDTTLRRSQMSPQGLRVRLRPGEERHFELEVFEPLESPVDLYILMDF SNSMSDDLDNLKKMGQNLARVLSQLTSDYTIGFGKFVDKVSVPQTDMRPEKLKEPWPNSDPPFSFKNVI SLTEDVDEFRNKLQGERISGNLDAPEGGFDAILQTAVCTRDIGWRPDSTHLLVFSTESAFHYEADGANV LAGIMSRNDERCHLDTTGTYTQYRTQDYPSVPTLVRLLAKHNIIPIFAVTNYSYSYYEKLHTYFPVSSL GVLQEDSSNIVELLEEAFNRIRSNLDIRALDSPRGLRTEVTSKMFQKTRTGSFHIRRGEVGIYQVQLRA LEHVDGTHVCQLPEDQKGNIHLKPSFSDGLKMDAGIICDVCTCELQKEVRSARCSFNGDFVCGQCVCSE GWSGQTCNCSTGSLSDIQPCLREGEDKPCSGRGECQCGHCVCYGEGRYEGQFCEYDNFQCPRTSGFLCN DRGRCSMGQCVCEPGWTGPSCDCPLSNATCIDSNGGICNGRGHCECGRCHCHQQSLYTDTICEINYSAI HPGLCEDLRSCVQCQAWGTGEKKGRTCEECNFKVKMVDELKRAEEVVVRCSFRDEDDDCTYSYTMEGDG APGPNSTVLVHKKKDCPPGSFWWLIPLLLLLLPLLALLLLLCWKYCACCKACLALLPCCNRGHMVGFKE DHYMLRENLMASDHLDTPMLRSGNLKGRDVVRWKVTNNMQRPGFATHAASINPTELVPYGLSLRLARLC TENLLKPDTRECAQLRQEVEENLNEVYRQISGVHKLQQTKFRQQPNAGKKQDHTIVDTVLMAPRSAKPA LLKLTEKQVEQRAFHDLKVAPGYYTLTADQDARGMVEFQEGVELVDVRVPLFIRPEDDDEKQLLVEAID VPAGTATLGRRLVNITIIKEQARDVVSFEQPEFSVSRGDQVARIPVIRRVLDGGKSQVSYRTQDGTAQG NRDYIPVEGELLFQPGEAWKELQVKLLELQEVDSLLRGRQVRRFHVQLSNPKFGAHLGQPHSTTIIIRD PDELDRSFTSQMLSSQPPPHGDLGAPQNPNAKAAGSRKIHFNWLPPSGKPMGYRVKYWIQGDSESEAHL LDSKVPSVELTNLYPYCDYEMKVCAYGAQGEGPYSSLVSCRTHQEVPSEPGRLAFNVVSSTVTQLSWAE PAETNGEITAYEVCYGLVNDDNRPIGPMKKVLVDNPKNRMLLIENLRESQPYRYTVKARNGAGWGPERE AIINLATQPKRPMSIPIIPDIPIVDAQSGEDYDSFLMYSDDVLRSPSGSQRPSVSDDTGCGWKFEPLLG EELDLRRVTWRLPPELIPRLSASSGRSSDAEAPHGPPDDGGAGGKGGSLPRSATPGPPGEHLVNGRMDF AFPGSTNSLHRMTTTSAAAYGTHLSPHVPHRVLSTSSTLTRDYNSLTRSEHSHSTTLPRDYSTLTSVSS HDSRLTAGVPDTPTRLVFSALGPTSLRVSWQEPRCERPLQGYSVEYQLLNGGELHRLNIPNPAQTSVVV EDLLPNHSYVFRVRAQSQEGWGREREGVITIESQVHPQSPLCPLPGSAFTLSTPSAPGPLVFTALSPDS LQLSWERPRRPNGDIVGYLVTCEMAQGGGPATAFRVDGDSPESRLTVPGLSENVPYKFKVQARTTEGFG PEREGIITIESQDGGPFPQLGSRAGLFQHPLQSEYSSITTTHTSATEPFLVDGLTLGAQHLEAGGSLTR HVTQEFVSRTLTTSGTLSTHMDQQFFQT As used herein, the term “collagen VII” has its general meaning in the art and refers to a type of collagen that forms anchoring fibrils that connect the epidermis and the dermis of the skin. 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. MTLRLLVAALCAGILAEAPRVRAQHRERVTCTRLYAADIVFLLDGSSSIGRSNFREVRSFLEGLVLPFS GAASAQGVRFATVQYSDDPRTEFGLDALGSGGDVIRAIRELSYKGGNTRTGAAILHVADHVFLPQLARP GVPKVCILITDGKSQDLVDTAAQRLKGQGVKLFAVGIKNADPEELKRVASQPTSDFFFFVNDFSILRTL LPLVSRRVCTTAGGVPVTRPPDDSTSAPRDLVLSEPSSQSLRVQWTAASGPVTGYKVQYTPLTGLGQPL PSERQEVNVPAGETSVRLRGLRPLTEYQVTVIALYANSIGEAVSGTARTTALEGPELTIQNTTAHSLLV AWRSVPGATGYRVTWRVLSGGPTQQQELGPGQGSVLLRDLEPGTDYEVTVSTLFGRSVGPATSLMARTD ASVEQTLRPVILGPTSILLSWNLVPEARGYRLEWRRETGLEPPQKVVLPSDVTRYQLDGLQPGTEYRLT LYTLLEGHEVATPATVVPTGPELPVSPVTDLQATELPGQRVRVSWSPVPGATQYRIIVRSTQGVERTLV LPGSQTAFDLDDVQAGLSYTVRVSARVGPREGSASVLTVRREPETPLAVPGLRVVVSDATRVRVAWGPV PGASGFRISWSTGSGPESSQTLPPDSTATDITGLQPGTTYQVAVSVLRGREEGPAAVIVARTDPLGPVR TVHVTQASSSSVTITWTRVPGATGYRVSWHSAHGPEKSQLVSGEATVAELDGLEPDTEYTVHVRAHVAG VDGPPASVVVRTAPEPVGRVSRLQILNASSDVLRITWVGVTGATAYRLAWGRSEGGPMRHQILPGNTDS AEIRGLEGGVSYSVRVTALVGDREGTPVSIVVTTPPEAPPALGTLHVVQRGEHSLRLRWEPVPRAQGFL LHWQPEGGQEQSRVLGPELSSYHLDGLEPATQYRVRLSVLGPAGEGPSAEVTARTESPRVPSIELRVVD TSIDSVTLAWTPVSRASSYILSWRPLRGPGQEVPGSPQTLPGISSSQRVTGLEPGVSYIFSLTPVLDGV RGPEASVTQTPVCPRGLADVVFLPHATQDNAHRAEATRRVLERLVLALGPLGPQAVQVGLLSYSHRPSP LFPLNGSHDLGIILQRIRDMPYMDPSGNNLGTAVVTAHRYMLAPDAPGRRQHVPGVMVLLVDEPLRGDI FSPIREAQASGLNVVMLGMAGADPEQLRRLAPGMDSVQTFFAVDDGPSLDQAVSGLATALCQASFTTQP RPEPCPVYCPKGQKGEPGEMGLRGQVGPPGDPGLPGRTGAPGPQGPPGSATAKGERGFPGADGRPGSPG RAGNPGTPGAPGLKGSPGLPGPRGDPGERGPRGPKGEPGAPGQVIGGEGPGLPGRKGDPGPSGPPGPRG PLGDPGPRGPPGLPGTAMKGDKGDRGERGPPGPGEGGIAPGEPGLPGLPGSPGPQGPVGPPGKKGEKGD SEDGAPGLPGQPGSPGEQGPRGPPGAIGPKGDRGFPGPLGEAGEKGERGPPGPAGSRGLPGVAGRPGAK GPEGPPGPTGRQGEKGEPGRPGDPAVVGPAVAGPKGEKGDVGPAGPRGATGVQGERGPPGLVLPGDPGP KGDPGDRGPIGLTGRAGPPGDSGPPGEKGDPGRPGPPGPVGPRGRDGEVGEKGDEGPPGDPGLPGKAGE RGLRGAPGVRGPVGEKGDQGDPGEDGRNGSPGSSGPKGDRGEPGPPGPPGRLVDTGPGAREKGEPGDRG QEGPRGPKGDPGLPGAPGERGIEGFRGPPGPQGDPGVRGPAGEKGDRGPPGLDGRSGLDGKPGAAGPSG PNGAAGKAGDPGRDGLPGLRGEQGLPGPSGPPGLPGKPGEDGKPGLNGKNGEPGDPGEDGRKGEKGDSG ASGREGRDGPKGERGAPGILGPQGPPGLPGPVGPPGQGFPGVPGGTGPKGDRGETGSKGEQGLPGERGL RGEPGSVPNVDRLLETAGIKASALREIVETWDESSGSFLPVPERRRGPKGDSGEQGPPGKEGPIGFPGE RGLKGDRGDPGPQGPPGLALGERGPPGPSGLAGEPGKPGIPGLPGRAGGVGEAGRPGERGERGEKGERG EQGRDGPPGLPGTPGPPGPPGPKVSVDEPGPGLSGEQGPPGLKGAKGEPGSNGDQGPKGDRGVPGIKGD RGEPGPRGQDGNPGLPGERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGE PGETGPPGRGLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVP GSPGLPGPVGPKGEPGPTGAPGQAVVGLPGAKGEKGAPGGLAGDLVGEPGAKGDRGLPGPRGEKGEAGR AGEPGDPGEDGQKGAPGPKGFKGDPGVGVPGSPGPPGPPGVKGDLGLPGLPGAPGVVGFPGQTGPRGEM GQPGPSGERGLAGPPGREGIPGPLGPPGPPGSVGPPGASGLKGDKGDPGVGLPGPRGERGEPGIRGEDG RPGQEGPRGLTGPPGSRGERGEKGDVGSAGLKGDKGDSAVILGPPGPRGAKGDMGERGPRGLDGDKGPR GDNGDPGDKGSKGEPGDKGSAGLPGLRGLLGPQGQPGAAGIPGDPGSPGKDGVPGIRGEKGDVGFMGPR GLKGERGVKGACGLDGEKGDKGEAGPPGRPGLAGHKGEMGEPGVPGQSGAPGKEGLIGPKGDRGFDGQP GPKGDQGEKGERGTPGIGGFPGPSGNDGSAGPPGPPGSVGPRGPEGLQGQKGERGPPGERVVGAPGVPG APGERGEQGRPGPAGPRGEKGEAALTEDDIRGFVRQEMSQHCACQGQFIASGSRPLPSYAADTAGSQLH AVPVLRVSHAEEEERVPPEDDEYSEYSEYSVEEYQDPEAPWDSDDPCSLPLDEGSCTAYTLRWYHRAVT GSTEACHPFVYGGCGGNANRFGTREACERRCPPRVVQSQGTGTAQD As used herein, the term “BP230” also known as Dystonin (DST) or bullous pemphigoid antigen 1 (BPAG1), refers to a cytoskeletal linker protein. 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). 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. SEQ ID NO:5 >sp|Q03001-3|DYST_HUMAN Isoform 3 of Dystonin OS=Homo sapiens OX=9606 GN=DST MHSSSYSYRSSDSVFSNTTSTRTSLDSNENLLLVHCGPTLINSCISFGSESFDGHRLEMLQQIANRVQR DSVICEDKLILAGNALQSDSKRLESGVQFQNEAEIAGYILECENLLRQHVIDVQILIDGKYYQADQLVQ RVAKLRDEIMALRNECSSVYSKGRILTTEQTKLMISGITQSLNSGFAQTLHPSLTSGLTQSLTPSLTSS SMTSGLSSGMTSRLTPSVTPAYTPGFPSGLVPNFSSGVEPNSLQTLKLMQIRKPLLKSSLLDQNLTEEE INMKFVQDLLNWVDEMQVQLDRTEWGSDLPSVESHLENHKNVHRAIEEFESSLKEAKISEIQMTAPLKL TYAEKLHRLESQYAKLLNTSRNQERHLDTLHNFVSRATNELIWLNEKEEEEVAYDWSERNTNIARKKDY HAELMRELDQKEENIKSVQEIAEQLLLENHPARLTIEAYRAAMQTQWSWILQLCQCVEQHIKENTAYFE FFNDAKEATDYLRNLKDAIQRKYSCDRSSSIHKLEDLVQESMEEKEELLQYKSTIANLMGKAKTIIQLK PRNSDCPLKTSIPIKAICDYRQIEITIYKDDECVLANNSHRAKWKVISPTGNEAMVPSVCFTVPPPNKE AVDLANRIEQQYQNVLTLWHESHINMKSVVSWHYLINEIDRIRASNVASIKTMLPGEHQQVLSNLQSRF EDFLEDSQESQVFSGSDITQLEKEVNVCKQYYQELLKSAEREEQEESVYNLYISEVRNIRLRLENCEDR LIRQIRTPLERDDLHESVFRITEQEKLKKELERLKDDLGTITNKCEEFFSQAAASSSVPTLRSELNVVL QNMNQVYSMSSTYIDKLKTVNLVLKNTQAAEALVKLYETKLCEEEAVIADKNNIENLISTLKQWRSEVD EKRQVFHALEDELQKAKAISDEMFKTYKERDLDFDWHKEKADQLVERWQNVHVQIDNRLRDLEGIGKSL KYYRDTYHPLDDWIQQVETTQRKIQENQPENSKTLATQLNQQKMLVSEIEMKQSKMDECQKYAEQYSAT VKDYELQTMTYRAMVDSQQKSPVKRRRMQSSADLIIQEFMDLRTRYTALVTLMTQYIKFAGDSLKRLEE EEIKRCKETSEHGAYSDLLQRQKATVLENSKLTGKISELERMVAELKKQKSRVEEELPKVREAAENELR KQQRNVEDISLQKIRAESEAKQYRRELETIVREKEAAERELERVRQLTIEAEAKRAAVEENLLNFRNQL EENTFTRRTLEDHLKRKDLSLNDLEQQKNKLMEELRRKRDNEEELLKLIKQMEKDLAFQKQVAEKQLKE KQKIELEARRKITEIQYTCRENALPVCPITQATSCRAVTGLQQEHDKQKAEELKQQVDELTAANRKAEQ DMRELTYELNALQLEKTSSEEKARLLKDKLDETNNTLRCLKLELERKDQAEKGYSQQLRELGRQLNQTT GKAEEAMQEASDLKKIKRNYQLELESLNHEKGKLQREVDRITRAHAVAEKNIQHLNSQIHSFRDEKELE RLQICQRKSDHLKEQFEKSHEQLLQNIKAEKENNDKIQRLNEELEKSNECAEMLKQKVEELTRQNNETK LMMQRIQAESENIVLEKQTIQQRCEALKIQADGFKDQLRSTNEHLHKQTKTEQDFQRKIKCLEEDLAKS QNLVSEFKQKCDQQNIIIQNTKKEVRNLNAELNASKEEKRRGEQKVQLQQAQVQELNNRLKKVQDELHL KTIEEQMTHRKMVLFQEESGKFKQSAEEFRKKMEKLMESKVITENDISGIRLDFVSLQQENSRAQENAK LCETNIKELERQLQQYREQMQQGQHMEANHYQKCQKLEDELIAQKREVENLKQKMDQQIKEHEHQLVLL QCEIQKKSTAKDCTFKPDFEMTVKECQHSGELSSRNTGHLHPTPRSPLLRWTQEPQPLEEKWQHRVVEQ IPKEVQFQPPGAPLEKEKSQQCYSEYFSQTSTELQITFDETNPITRLSEIEKIRDQALNNSRPPVRYQD NACEMELVKVLTPLEIAKNKQYDMHTEVTTLKQEKNPVPSAEEWMLEGCRASGGLKKGDFLKKGLEPET FQNFDGDHACSVRDDEFKFQGLRHTVTARQLVEAKLLDMRTIEQLRLGLKTVEEVQKTLNKFLTKATSI AGLYLESTKEKISFASAAERIIIDKMVALAFLEAQAATGFIIDPISGQTYSVEDAVLKGVVDPEFRIRL LEAEKAAVGYSYSSKTLSVFQAMENRMLDRQKGKHILEAQIASGGVIDPVRGIRVPPEIALQQGLLNNA ILQFLHEPSSNTRVFPNPNNKQALYYSELLRMCVFDVESQCFLFPFGERNISNLNVKKTHRISVVDTKT GSELTVYEAFQRNLIEKSIYLELSGQQYQWKEAMFFESYGHSSHMLTDTKTGLHFNINEAIEQGTIDKA LVKKYQEGLITLTELADSLLSRLVPKKDLHSPVAGYWLTASGERISVLKASRRNLVDRITALRCLEAQV STGGIIDPLTGKKYRVAEALHRGLVDEGFAQQLRQCELVITGIGHPITNKMMSVVEAVNANIINKEMGI RCLEFQYLTGGLIEPQVHSRLSIEEALQVGIIDVLIATKLKDQKSYVRNIICPQTKRKLTYKEALEKAD FDFHTGLKLLEVSEPLMTGISSLYYSS As used herein, the term “Laminin” has its general meaning in the art and refers to a family of glycoproteins of the extracellular matrix of all animals. They 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 (α, β, and γ) 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. These glycoproteins are imperative to the maintenance and vitality of tissues; defective laminins can cause muscles to form improperly, leading to for example a form of lethal skin blistering disease (junctional epidermolysis bullosa). 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. sapiens OX=9606 GN=LAMB4 PE=1 SV=1 MQFQLTLFLHLGWLSYSKAQDDCNRGACHPTTGDLLVGRNTQLMASSTCGLSRAQKYCILSYLEGEQKC FICDSRFPYDPYDQPNSHTIENVIVSFEPDREKKWWQSENGLDHVSIRLDLEALFRFSHLILTFKTFRP AAMLVERSTDYGHNWKVFKYFAKDCATSFPNITSGQAQGVGDIVCDSKYSDIEPSTGGEVVLKVLDPSF EIENPYSPYIQDLVTLTNLRINFTKLHTLGDALLGRRQNDSLDKYYYALYEMIVRGSCFCNGHASECRP MQKMRGDVFSPPGMVHGQCVCQHNTDGPNCERCKDFFQDAPWRPAADLQDNACRSCSCNSHSSRCHFDM TTYLASGGLSGGVCEDCQHNTEGQHCDRCRPLFYRDPLKTISDPYACIPCECDPDGTISGGICVSHSDP ALGSVAGQCLCKENVEGAKCDQCKPNHYGLSATDPLGCQPCDCNPLGSLPFLTCDVDTGQCLCLSYVTG AHCEECTVGYWGLGNHLHGCSPCDCDIGGAYSNVCSPKNGQCECRPHVTGRSCSEPAPGYFFAPLNFYL YEAEEATTLQGLAPLGSETFGQSPAVHVVLGEPVPGNPVTWTGPGFARVLPGAGLRFAVNNIPFPVDFT IAIHYETQSAADWTVQIVVNPPGGSEHCIPKTLQSKPQSFALPAATRIMLLPTPICLEPDVQYSIDVYF SQPLQGESHAHSHVLVDSLGLIPQINSLENFCSKQDLDEYQLHNCVEIASAMGPQVLPGACERLIISMS AKLHDGAVACKCHPQGSVGSSCSRLGGQCQCKPLVVGRCCDRCSTGSYDLGHHGCHPCHCHPQGSKDTV CDQVTGQCPCHGEVSGRRCDRCLAGYFGFPSCHPCPCNRFAELCDPETGSCFNCGGFTTGRNCERCIDG YYGNPSSGQPCRPCLCPDDPSSNQYFAHSCYQNLWSSDVICNCLQGYTGTQCGECSTGFYGNPRISGAP CQPCACNNNIDVTDPESCSRVTGECLRCLHNTQGANCQLCKPGHYGSALNQTCRRCSCHASGVSPMECP PGGGACLCDPVTGACPCLPNVTGLACDRCADGYWNLVPGRGCQSCDCDPRTSQSSHCDQLTGQCPCKLG YGGKRCSECQENYYGDPPGRCIPCDCNRAGTQKPICDPDTGMCRCREGVSGQRCDRCARGHSQEFPTCL QCHLCFDQWDHTISSLSKAVQGLMRLAANMEDKRETLPVCEADFKDLRGNVSEIERILKHPVFPSGKFL KVKDYHDSVRRQIMQLNEQLKAVYEFQDLKDTIERAKNEADLLLEDLQEEIDLQSSVLNASIADSSENI KKYYHISSSAEKKINETSSTINTSANTRNDLLTILDTLTSKGNLSLERLKQIKIPDIQILNEKVCGDPG NVPCVPLPCGGALCTGRKGHRKCRGPGCHGSLTLSTNALQKAQEAKSIIRNLDKQVRGLKNQIESISEQ AEVSKNNALQLREKLGNIRNQSDSEEENINLFIKKVKNFLLEENVPPEDIEKVANGVLDIHLPIPSQNL TDELVKIQKHMQLCEDYRTDENRLNEEADGAQKLLVKAKAAEKAANILLNLDKTLNQLQQAQITQGRAN STITQLTANITKIKKNVLQAENQTREMKSELELAKQRSGLEDGLSLLQTKLQRHQDHAVNAKVQAESAQ HQAGSLEKEFVELKKQYAILQRKTSTTGLTKETLGKVKQLKDAAEKLAGDTEAKIRRITDLERKIQDLN LSRQAKADQLRILEDQVVAIKNEIVEQEKKYARCYS MRGSHRAAPALRPRGRLWPVLAVLAAAAAAGCAQAAMDECTDEGGRPQRCMPEFVNAAFNVTVVATNTC GTPPEEYCVQTGVTGVTKSCHLCDAGQPHLQHGAAFLTDYNNQADTTWWQSQTMLAGVQYPSSINLTLH LGKAFDITYVRLKFHTSRPESFAIYKRTREDGPWIPYQYYSGSCENTYSKANRGFIRTGGDEQQALCTD EFSDISPLTGGNVAFSTLEGRPSAYNFDNSPVLQEWVTATDIRVTLNRLNTFGDEVFNDPKVLKSYYYA ISDFAVGGRCKCNGHASECMKNEFDKLVCNCKHNTYGVDCEKCLPFFNDRPWRRATAESASECLPCDCN GRSQECYFDPELYRSTGHGGHCTNCQDNTDGAHCERCRENFFRLGNNEACSSCHCSPVGSLSTQCDSYG RCSCKPGVMGDKCDRCQPGFHSLTEAGCRPCSCDPSGSIDECNIETGRCVCKDNVEGFNCERCKPGFFN LESSNPRGCTPCFCFGHSSVCTNAVGYSVYSISSTFQIDEDGWRAEQRDGSEASLEWSSERQDIAVISD SYFPRYFIAPAKFLGKQVLSYGQNLSFSFRVDRRDTRLSAEDLVLEGAGLRVSVPLIAQGNSYPSETTV KYVFRLHEATDYPWRPALTPFEFQKLLNNLTSIKIRGTYSERSAGYLDDVTLASARPGPGVPATWVESC TCPVGYGGQFCEMCLSGYRRETPNLGPYSPCVLCACNGHSETCDPETGVCNCRDNTAGPHCEKCSDGYY GDSTAGTSSDCQPCPCPGGSSCAVVPKTKEVVCTNCPTGTTGKRCELCDDGYFGDPLGRNGPVRLCRLC QCSDNIDPNAVGNCNRLTGECLKCIYNTAGFYCDRCKDGFFGNPLAPNPADKCKACNCNLYGTMKQQSS CNPVTGQCECLPHVTGQDCGACDPGFYNLQSGQGCERCDCHALGSTNGQCDIRTGQCECQPGITGQHCE RCEVNHFGFGPEGCKPCDCHPEGSLSLQCKDDGRCECREGFVGNRCDQCEENYFYNRSWPGCQECPACY RLVKDKVADHRVKLQELESLIANLGTGDEMVTDQAFEDRLKEAEREVMDLLREAQDVKDVDQNLMDRLQ RVNNTLSSQISRLQNIRNTIEETGNLAEQARAHVENTERLIEIASRELEKAKVAAANVSVTQPESTGDP NNMTLLAEEARKLAERHKQEADDIVRVAKTANDTSTEAYNLLLRTLAGENQTAFEIEELNRKYEQAKNI SQDLEKQAARVHEEAKRAGDKAVEIYASVAQLSPLDSETLENEANNIKMEAENLEQLIDQKLKDYEDLR EDMRGKELEVKNLLEKGKTEQQTADQLLARADAAKALAEEAAKKGRDTLQEANDILNNLKDFDRRVNDN KTAAEEALRKIPAINQTITEANEKTREAQQALGSAAADATEAKNKAHEAERIASAVQKNATSTKAEAER TFAEVTDLDNEVNNMLKQLQEAEKELKRKQDDADQDMMMAGMASQAAQEAEINARKAKNSVTSLLSIIN DLLEQLGQLDTVDLNKLNEIEGTLNKAKDEMKVSDLDRKVSDLENEAKKQEAAIMDYNRDIEEIMKDIR NLEDIRKTLPSGCFNTPSIEKP Methods of the present invention: The first object of the present invention relates to a method for detecting the presence of autoantibodies associated autoimmune bullous skin diseases in sample obtained from a subject comprising the steps of: - a) placing a sample obtained from the subject, in a single assay receptacle, in the presence of one or more particles selected from the group consisting of particles being conjugated to the NC16A domain of BP180, particles being conjugated to the C-terminal domain of BP180, particles being conjugated to the LAD-1 domains of BP180, particles conjugates to the integrin α6β4, particles being conjugated to the NC1 domain of collagen VII, particles being conjugated to the NC2 domain of collagen VII, particles being conjugated to the BP230, particles being conjugated to the γ1 chains of P200 laminin and / or particles being conjugated to the β4 chains of P200 laminin. - b) incubating the mixture under conditions which allow the formation of immunocomplexes on particles, - c) eliminating the immunoglobulins which have not bound to the particles, and - d) detecting the immunocomplexes of step b) on the plurality of particles, whereby the presence or absence of autoantibodies directed against the NC16A domain of BP180, the C-terminal domain of BP180, the LAD-1 domains of BP180, the integrin α6β4, the NC1 domain of collagen VII, the NC2 domain of collagen VII, the BP230, the γ1 chains of P200 laminin or the β4 chains of P200 laminin is revealed. In some embodiments, 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 methacrylate), copolymers styrene / butadiene, styrene / acrylic acid, styrene / vinylpyrrolidone and butadiene / acrylonitrile, or methoxy poly(ethylene glycol)-poly(lactide) copolymer (MPEG-PLA). Polymer particles can be crosslinked or not. For instance, 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 (polytetrafluoroethylene). In some embodiments, 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. In some embodiments, the particles are magnetic and coded. In particular, 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. With regard to coded particles, each particle can include a unique code, preferably, 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. For example, 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. For example, Chandler et al. (U.S. Pat. No. 5,981,180) describes a particle-based system in which different particle types are encoded by mixtures of various proportions of two or more fluorescent dyes impregnated into polymer particles. Soini (U.S. Pat. No. 5,028,545) describes a particle-based multiplexed assay system that employs time- resolved fluorescence for particle identification. 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. As used herein, the term “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 Fe3O4, BaFe12O19, Mn2O3, Cr2O3, CoO, NiO, and CoMnP. In some embodiments, 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. Preferably 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). Exemplary methods for the preparation of or composition of magnetic particles are described in, e.g., U.S. Pat. Nos.6,773,812 and 6,280,618. In some embodiments, a first group of particles is conjugated to a polypeptide that derives from the NC16A domain of BP180. In some embodiments, 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. In some embodiments, 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. In some embodiments, a second group of particles is conjugated to polypeptide that derives from the C-terminal domain of BP180. In some embodiments, 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. In some embodiments, 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. In some embodiments, a third group of particles is conjugated to a first polypeptide that derives from the alpha 6 subunit of the integrin α6β4 and / or to a second polypeptide that derives from the beta 4 subunit of the integrin α6β4. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, a fourth group of particles is conjugated to a polypeptide that derives from the NC1 domain of COL7A1. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, an eighth group of particles is conjugated to a polypeptide that derived from the β4 chain of P200 laminin. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, a nineth group of particles is conjugated to a polypeptide that derived from the γ1 chain of P200 laminin. In some embodiments, 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. In some embodiments, 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. In some embodiments, the polypeptides that are conjugated to the particles can comprise a tag. For example, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. For example, 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. In some embodiments, the polypeptides are conjugated directly to the particle, meaning that they form a covalent bond with a reactive group on the particle surface. In some embodiments, 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. For example, 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. In some embodiments, 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. In some embodiments, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC)- N- hydroxysulfosuccinimide (Sulfo NHS) reactions are used for conjugating the polypeptides to the particles. In some embodiments, 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. Heterobifunctional reagents are preferred. A typical bifunctional cross-linking agent is N-succinimidyl(4-iodoacetyl) aminobenzoate (SIAB). However, other crosslinking agents, including, without limitation, dimaleimide, dithio-bis-nitrobenzoic acid (DTNB), N- succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and 6- hydrazinonicotimide (HYNIC) may also be used. For further examples of 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). In some embodiments, 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 β4 chain of P200 laminin and the γ1 chain of P200 laminin respectively, by covalent bonds between a reactive group on the particle surface and an amino acid residue on the polypeptide. In some embodiments, the polypeptide that derives from the NC2 domain of COL7A1 is indirectly conjugated to the particles belonging to the fifth group as described above. In some embodiments, 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. In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody that specifically binds to the tag. In some embodiments, 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. In some embodiments, the receptacle may be any solid container, for example a test tube, a microplate well or a reaction cuvette made of polypropylene. In some embodiments, 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. As used herein the term “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. In some embodiments, the method of the present invention includes use of a secondary antibody that is coupled to an indicator reagent comprising a signal generating compound. In some embodiments, the secondary antibody has specificity for a particular immunoglobulin. In some embodiments, the secondary antibody is an anti-human IgG antibody, including anti- IgG1, IgG2, IgG3 and IgG4 antibodies. In some embodiments, the secondary antibody is an anti-human IgA antibody, in particular an anti-IgA1 or IgA2 antibody. In some embodiments, the secondary antibody is an anti-human IgE antibody. In some embodiments, the antibody having specificity for a particular type immunoglobulin is a mouse, rabbit or goat antibody. In some embodiments, 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. Examples of enzyme conjugates include alkaline phosphatase, horseradish peroxidase and beta-galactosidase. In some embodiments, the secondary antibody is conjugated to phycoerythrin. Methods for detecting the particle identity codes, e.g., a fluorescent code, 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. Descriptions of instrumentation and methods for flow cytometry are known in the art and include, e.g., McHugh, “Flow Microsphere Immunoassay for the Quantitative and Simultaneous Detection of Multiple Soluble Analytes,” Methods in Cell Biology 42, Part B (Academic Press, 1994); McHugh et al., “Microsphere-Based Fluorescence Immunoassays Using Flow Cytometry Instrumentation,” Clinical Flow Cytometry, Bauer, K. D., et al., eds. (Baltimore, Md., USA: Williams and Williams, 1993), pp.535-544; Lindmo et al, “Immunometric Assay Using Mixtures of Two Particle Types of Different Affinity,” J. Immunol. Meth. 126: 183-189 (1990); McHugh, “Flow Cytometry and the Application of Microsphere-Based Fluorescence Immunoassays,” Immunochemica 5: 116 (1991); Horan et al., “Fluid Phase Particle Fluorescence Analysis: Rheumatoid Factor Specificity Evaluated by Laser Flow Cytophotometry,” Immunoassays in the Clinical Laboratory, 185-189 (Liss 1979); Wilson et al, “A New Microsphere-Based Immunofluorescence Assay Using Flow Cytometry,” J. Immunol. Meth.107: 225-230 (1988); Fulwyler et al., “Flow Microsphere Immunoassay for the Quantitative and Simultaneous Detection of Multiple Soluble Analytes,” Meth. Cell Biol. 33: 613-629 (1990); Coulter Electronics Inc., United Kingdom Patent No.1,561,042 (published Feb. 13, 1980); and Steinkamp et al., Review of Scientific Instruments 44(9): 1301-1310 (1973). Typically, the detecting step thus involved the use of detector. As used herein, the term “detector” is intended to mean a device or apparatus that converts the energy of contacted photons into an electrical response. For instance, 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. In particular, the detector involves the use of a radiation source. As used herein, 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. As used herein, 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. These and other lasers useful in the apparatus of the invention are known in the art as described, for example, in Shapiro, Practical Flow Cytometry, 3rd Ed. Wiley-Liss, New York (1995). In some embodiments, the detector is a flow cytometer. As used herein, the term “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. In particular, 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 α6β4, the NC1 domain of collagen VII, the NC2 domain of collagen VII, the LAD-1 domain of BP180, the BP230, the β4 chain of P200 laminin or the γ1 chain of P200 laminin in a single sample. In some embodiments, the groups of said particles differ from one another by their identity codes (e.g. fluorophores) as described above. In said embodiments, the method of the present invention thus involves the use of a multiplex technology. As used herein, 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. In some embodiments, the method of the present invention involves an addressable laser bead immunoassay (ALBIA), which is commercially available on Luminex™-based platforms. For instance, 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 α6β4, the NC1 domain of collagen VII, the NC2 domain of collagen VII, the LAD-1 domain of BP180, the BP230, the β4 chain of P200 laminin and the γ1 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). After washing again, 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. It should be noted that 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. In some embodiments, 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. As used herein the term “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. As used herein the term “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). In particular, 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. As used herein, 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. As used herein, the term "relapse" refers to the return of signs and symptoms of a disease after a subject has enjoyed a remission after a treatment. Thus, if initially 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." Typically, the treatment is an immunosuppressive treatment. According to the present invention, the treatment consists in any method or drug that could be suitable for the treatment of an autoimmune bullous skin disease. In some embodiments, the treatment consists in an antibody depleting strategy, which typically include plasma exchange, plasmapheresis or immunoadsorption. In some embodiments, the treatment consists in administering immunoglobulins (e.g. by intravenous route). In some embodiments, the treatment is an immunosuppressive treatment. As used herein, the term “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. In some embodiments, the immunosuppressive drug is a corticosteroid. As used, the term “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 (11β,17-dihydroxy, 21-(phosphonooxy)-pregn-4-ene, 3,20-dione disodium), dihydroxycortisone, dexamethasone (21-(acetyloxy)-9-fluoro-1β,17-dihydroxy-16α-m- ethylpregna-1,4-diene-3,20-dione), and highly derivatized steroid drugs such as beconase (beclomethasone dipropionate, which is 9-chloro-11-β, 17,21, trihydroxy-16β-methylpregna- 1,4 diene-3,20-dione 17,21-dipropionate). Other examples of 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. In some embodiments, the treatment consists of administering a B cell depleting agent. As used herein, the term “B cell depleting agent” refers to any agent that is capable of triggering lymphodepletion of B cells. In some embodiments, 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. 5,736,137, expressly incorporated herein by reference), a chimeric pan-B antibody targeting CD20; the yttrium-

[0090] -labeled 2B8 murine antibody designated “Y2B8” or “Ibritumomab Tiuxetan” ZEVALIN® (U.S. Pat. No.5,736,137, expressly incorporated herein by reference), a murine IgG1 kappa mAb covalently linked to MX-DTPA for chelating to yttrium-

[0090] ; murine IgG2a “BI,” also called “Tositumomab,” optionally labeled with radioactive 131I to generate the “1311-B1” antibody (iodine 131 tositumomab, BEXXAR™) (U.S. Pat. No. 5,595,721, expressly incorporated herein by reference); murine monoclonal antibody “1F5” (Press et al. Blood 69 (2):584-591 (1987) and variants thereof including “framework patched” or humanized 1F5 (WO03 / 002607, Leung, S.; ATCC deposit HB- 96450); murine 2H7 and chimeric 2H7 antibody (U.S. Pat. No. 5,677,180, expressly incorporated herein by reference); humanized 2H7, also known as ocrelizumab (PRO-70769); Ofatumumab (Arzerra), a fully human IgG1 against a novel epitope on CD20 huMax-CD20 (Genmab, Denmark; WO2004 / 035607 (U.S. Ser. No. 10 / 687,799, expressly incorporated herein by reference)); AME-133 (ocaratuzumab; Applied Molecular Evolution), a a fully- humanized and optimized IgG1 mAb against CD20; A20 antibody or variants thereof such as chimeric or humanized A20 antibody (cA20, hA20, respectively) (U.S. Ser. No. 10 / 366,709, expressly incorporated herein by reference, Immunomedics); and monoclonal antibodies L27, G28-2, 93-1B3, B-CI or NU-B2 available from the International Leukocyte Typing Workshop (Valentine et al, In: Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University Press (1987)). Further, suitable antibodies include e.g. antibody GA101 (obinutuzumab), a third generation humanized anti-CD20-antibody of Biogen Idec / Genentech / Roche. Moreover, 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. Further suitable antibodies are 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), a biosimilar version of rituximab developed by Intas Biopharmaceuticals, CT-P10), a biosimilar version of rituximab developed by Celltrion), a biosimilar version of rituximab developed by Biocad, Ublituximab (LFB-R603, a transgenically produced mAb targeting CD20 developed by GTC Biotherapeutics (LFB Biotechnologies)), PF-05280586 (presumed to be a biosimilar version of rituximab developed by Pfizer), Lymphomun (Bi-20, a trifunctional anti-CD20 and anti-CD3 antibody, developed by Trion Pharma), a biosimilar version of rituximab developed by Natco Pharma, a biosimilar version of rituximab developed by iBio, a biosimilar version of rituximab developed by Gedeon Richter / Stada, a biosimilar version of rituximab developed by Curaxys, a biosimilar version of rituximab developed by Coherus Biosciences / Daiichi Sankyo, a biosimilar version of rituximab developed by BioXpress, BT-D004 (a biosimilar version of rituximab developed by Protheon), AP-052 (a biosimilar version of rituximab developed by Aprogen), a biosimilar version of ofatumumab developed by BioXpress, MG-1106 (a biosimilar version of rituximab developed by Green Cross), IBI-301 (a humanized monoclonal antibody against CD20 developed by Innovent Biologics), BVX-20 (a humanized mAb against the CD20 developed by Vaccinex), 20-C2-2b (a bispecific mAb-IFNalpha that targets CD20 and human leukocyte antigen-DR (HLA-DR) developed by Immunomedics), MEDI-552 (developed by MedImmune / AstraZeneca), the anti-CD20 / streptavidin conjugates developed by NeoRx (now Poniard Pharmaceuticals), the 2nd generation anti-CD20 human antibodies developed by Favrille (now MMRGlobal), TRU-015, an antibody specific for CD20 fragment developed by Trubion / Emergent BioSolutions, as well as other preclinical approaches by various companies and entities. All aforementioned publications, references, patents and patent applications are incorporated by reference in their entireties. All antibodies disclosed in therein may be used within the present invention. Kits of the present invention: A further object of the present invention relates to a kit for performing the method of the present invention. In particular, 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 α6β4, NC1 domain of collagen VII, NC2 domain of collagen VII, BP230, γ1 chain of P200 laminin and β4 chain of P200 laminin; and means for detecting the binding of autoantibodies to the particles. 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 α6β4, NC1 domain of collagen VII, NC2 domain of collagen VII, BP230, γ1 chain of P200 laminin and / or β4 chain of P200 laminin; and means for detecting the binding of autoantibodies to the particles. 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. Thus, 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. In some embodiments, 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. In addition, the 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). α6β4 Integrin coupled beads were validated with anti-α6 (I) or anti-β4 antibodies (J). The γ1 chain of P200 laminin coupled beads were validated with anti- γ1 (K) or anti-DDK Tag antibodies (L). The β4 chain of P200 laminin coupled beads were validated with anti-β4 (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. Firstly, the beads coupled to anti-His Tag antibodies were validated with anti-mouse IgG antibody (Q). Secondly, the secondary coating of anti-His Tag beads with the NC2 domain of collagen VII was validated with anti-NC2 antibody (R). Finally, 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. Validation of serum dilution for detection of IgG directed against the nine proteins coupled beads (NC16A, C-terminal and LAD-1 domains of BP180, BP230, α6β4 Integrin, NC1 and NC2 domains of collagen VII and γ1 and β4 chains of P200 laminin).The 1 / 100 dilution was optimal for all bead-coupled proteins, allowing multiplexing. Figure 3: A-D. Validation of serum dilution for detection of IgA directed against the nine proteins coupled beads (NC16A, C-terminal and LAD-1 domains of BP180, BP230, α6β4 Integrin, NC1 and NC2 domains of collagen VII and γ1 and β4 chains of P200 laminin). The 1 / 100 dilution was optimal for all bead-coupled proteins, allowing multiplexing. Figure 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. Screening of 100 sera from healthy donors (HD), 80 sera from bullous pemphigoid (BP) patients, 33 from mucous membrane pemphigoid (MMP), 13 from epidermolysis bullosa acquisita (EBA) and 64 from P200 pemphigoid (P200) for the detection of IgG directed against nine dermal-epidermal junction proteins: BP180 (NC16A, C-terminal (C-ter), and LAD-1 domains), BP230, α6β4 integrin, type VII collagen (NC1 and NC2 domains), and P200 laminin (γ1 and β4 chains), using ALBIA (Adressable laser beads immunoassay). The cut-off values are indicated as horizontal dotted lines and determined by the mean + 2 standard deviations of HD. Mean values are shown as solid bars (SEM). Figure 7: Analysis of patients' sera using ALBIA to detect IgA directed against nine proteins of the dermal-epidermal junction. Screening of 100 sera from healthy donors (HD), 80 sera from bullous pemphigoid (BP) patients, 33 from mucous membrane pemphigoid (MMP), 13 from epidermolysis bullosa acquisita (EBA) and 64 from P200 pemphigoid (P200) for the detection of IgA directed against nine dermal-epidermal junction proteins: BP180 (NC16A, C-terminal (C-ter), and LAD-1 domains), BP230, α6β4 integrin, type VII collagen (NC1 and NC2 domains), and P200 laminin (γ1 and β4 chains), using ALBIA (Adressable laser beads immunoassay). The cut-off values are indicated as horizontal dotted lines and determined by the mean + 2 standard deviations of HD. Mean values are shown as solid bars (SEM). EXAMPLE: Methods: Human recombinant proteins from the dermo-epidermal junction. Nine human recombinant proteins from the DEJ were selected and were custom-made: NC16A, C-terminal and LAD-1 domains of BP180, BP230, α6β4 Integrin, NC1 and NC2 domains of collagen VII, and γ1 and β4 chains of P200 laminin. The characteristics of these nine human recombinant proteins were summarized in Table 1. 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 Integrin α6β4α6: AA 24-878His 115 Mammal cells β4: AA 28-710 76 BP180 LAD-1 AA 489-1497 His 113 Eukaryotic cells P200 laminin - γ1 AA 1-1609 DDK 178 Mammal cells P200 laminin – β4 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. For that, 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. It consisted of coupling the following human recombinant proteins: NC16A, C-terminal and LAD-1 domains of BP180, BP230, α6β4 Integrin, NC1 domain of collagen VII and γ1 and β4 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). The validation of serum dilution for detection of IgG (Figures 2A to 2D) and IgA (Figures 3A to 3D) directed against the nine human recombinant proteins were previously realized. To quantify the auto-antibodies, 1000 coated beads for each nine proteins were incubated in Multiscreen 96-well plates with 100µl of sera diluted at 1:100 in Dulbecco’s phosphate-buffered saline (DPBS) with Ca2+ / Mg2+and 1% fetal bovine serum for 1h30 on a plate shaker and then washed. Then, beads were incubated for 45min in the same conditions with 100µl 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 50µL of streptavidin-R-phycoerythrin (Biorad) diluted at 1:400, and 50µl of DPBS (without Ca2+ / Mg2+) 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. 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: (MFIserum / MFIpositivitythreshold) 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). Results: Finally, we analysed 80 sera from BP patients, 33 MMP, 13 EBA and 64 from P200 pemphigoid, using ALBIA to detect IgG (Figure 6) and IgA (Figure 7) directed against the nine proteins of the DEJ. Compared to currently available assays, the specificities and sensitivities were 94% and 80% for the detection of anti-NC16A antibodies (Data not shown), 93% and 88% for antibodies against the C-terminal and / or LAD-1 domains of BP180 (Data not shown), 93% and 75% for antibodies against BP230 (Data not shown), 99% and 84% for antibodies against NC1 domain of type VII collagen (Data not shown), and 98% and 88% for antibodies against β4 chain of P200 laminin (Data not shown). In addition, ALBIA detected IgG reactivity against BP180, BP230, and / or β4 P200 in 20 out of 23 (87%) seronegative PB patients (Data not shown). Overall, 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). Moreover, 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). Finally, ALBIA detected IgG reactivity against targeting at least one of the nine DEJ proteins in 57 out of 79 (72%) seronegative MMP patients (Data not shown). Overall, IgG and / or IgA targeting at least one of the nine DEJ proteins were identified in 73 out of 79 (92%) seronegative MMP patients (Data not shown). Overall, among the 112 seronegative patients with an unclear diagnosis, ALBIA IgG analysis identified at least one autoantibody target in 86 cases (77%). Combining IgG with IgA analysis increased the detection rate to 101 out of 112 patients (90%). In conclusion, the multiplex assay may help characterize autoantibody targets in various DEJ diseases, thereby reducing the number of patients with an unclear diagnosis. REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method for detecting the presence of autoantibodies associated autoimmune bullous skin diseases in sample obtained from a subject comprising the steps of: a) placing a sample obtained from the subject, in a single assay receptacle, in the presence of one or more particles selected from the group consisting of particles being conjugated to the NC16A domain of BP180, particles being conjugated to the C- terminal domain of BP180, particles being conjugated to the LAD-1 domains of BP180, particles conjugates to the integrin α6β4, particles being conjugated to the NC1 domain of collagen VII, particles being conjugated to the NC2 domain of collagen VII, particles being conjugated to the BP230, particles being conjugated to the γ1 chains of P200 laminin and / or particles being conjugated to the β4 chains of P200 laminin. b) incubating the mixture under conditions which allow the formation of immunocomplexes on particles, c) eliminating the immunoglobulins which have not bound to the particles, and d) detecting the immunocomplexes of step b) on the plurality of particles, whereby the presence or absence of autoantibodies directed against the NC16A domain of BP180, the C-terminal domain of BP180, the LAD-1 domains of BP180, the integrin α6β4, the NC1 domain of collagen VII, the NC2 domain of collagen VII, the BP230, the γ1 chains of P200 laminin or the β4 chains of P200 laminin is revealed.

2. The method according to claim 1 wherein a 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 489 to the amino acid residue at position 566 in SEQ ID NO:

1.

3. The method according to claim 1 wherein a 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.

4. The method according to claim 1 wherein a third group of particles is conjugated to a first polypeptide having an amino acid sequence having at least 90% of identity withthe 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 / or 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.

5. The method according to claim 1 wherein a 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.

6. The method according to claim 1 wherein a 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.

7. The method according to claim 1 wherein a 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.

8. The method according to claim 1 wherein a 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.

9. The method according to claim 1 wherein 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.

10. The method according to claim 1 wherein 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.

11. The method according to any one of claims 1 to 10 wherein the conjugated polypeptides comprise a tag.

12. The method according to any one of claims 1 to 11 wherein the polypeptides are directly or indirectly conjugated to the particles.

13. The method according to any one of claims 1 to 11 wherein the particles are magnetic and coded.

14. The method according to any one of claims 1 to 13 that includes use of a secondary antibody has specificity for a particular immunoglobulin such as IgG, IgA, or IgE.

15. Use of the method according to any one of claims 1 to 14 for the diagnosis of autoimmune bullous skin diseases.

16. Use of the method according to any one of claims 1 to 14 for determining whether a subject suffering from an autoimmune bullous skin disease achieves a response with a treatment.

17. Use of the method according to any one of claims 1 to 14 for determining whether a subject is at risk of relapse after a treatment.

18. 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 domains of BP180, integrin α6β4, NC1 domain of collagen VII, NC2 domain of collagen VI, BP230, γ1 chains of P200 laminin and β4 chains of P200 laminin; and means for detecting the binding of autoantibodies to the particles.

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