Autoantibody Detection for Diagnosis in Direct Immunofluorescence-Negative Mucous Membrane Pemphigoid

Ocular and Other Sites Compared


      To assess whether a panel of serum pemphigoid autoantibody tests could be used to confirm an immunopathologic diagnosis of mucous membrane pemphigoid (MMP) in direct immunofluorescent negative (DIF–) MMP patients.


      Prospective cross-sectional study.


      Seventy-six patients with multisite MMP with 45 matched control participants.


      Enzyme-linked immunosorbent assays (ELISAs) for BP180 and BP230 (MBL International), immunoglobulin A (IgA) A and immunoglobulin G indirect immunofluorescence (IIF) on human salt-split skin and the keratinocyte footprint assay for anti–laminin 332 antibodies.

      Main Outcome Measures

      Sensitivity and specificity of autoantibody detection and significant differences for individual tests and test combinations for MMP involving different sites.


      All DIF– patients (24/73 [31.8%]) had either ocular-only disease or ocular involvement in multisite disease. Serum pemphigoid autoantibodies were detected in 29 of 76 MMP patients (38.2%) compared with 3 of 45 control participants (6.7%). Autoantibody reactivity detected by any 1 or more of the tests was present in 6 of 24 DIF– patients (25%) compared with 22 of 49 DIF positive (DIF+) patients (44.9%). Ocular-only MMP serum reactivity was not significantly different for any test or test combination compared with control participants, whereas DIF– multisite ocular MMP differed for 1 ELISA and 3 of 7 test combinations. By contrast, for DIF+ nonocular MMP patients, all the individual tests, apart from IgA IIF, and all test combinations were significantly different compared with those for control participants. For the entire MMP cohort, the sensitivity of all individual tests was low, having a maximum of 21.05% for BP180 reactivity but increasing to 38.16% for an optimal test combination. Disease activity was associated strongly with positive serologic findings.


      Pemphigoid serum autoantibody tests did not provide immunopathologic evidence of MMP in ocular-only MMP patients but showed limited value in DIF– multisite ocular MMP patients. The requirement for immunopathologic confirmation of MMP by autoantibody detection is inappropriate for DIF– ocular-only MMP patients, resulting in missed diagnoses, delayed therapy, and poor outcomes. Alternative diagnostic criteria for ocular-only MMP are required to exclude the other causes of scarring conjunctivitis until more sensitive and specific immunopathologic tests become available.


      Abbreviations and Acronyms:

      BM (basement membrane), DIF (direct immunofluorescence), DIF+ (direct immunofluorescent positive), DIF– (direct immunofluorescent negative), ELISA (enzyme-linked immunosorbent assay), IIF (indirect immunofluorescence), IgA (immunoglobulin A), IgG (immunoglobulin G), MMP (mucous membrane pemphigoid), SSS (salt-split skin)
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        • Chan L.S.
        • Ahmed A.R.
        • Anhalt G.J.
        • et al.
        The first international consensus on mucous membrane pemphigoid: definition, diagnostic criteria, pathogenic factors, medical treatment, and prognostic indicators.
        Arch Dermatol. 2002; 138: 370-379
        • Jonkman M.F.
        • Groot A.C.
        • Slegers T.P.
        • et al.
        Immune diagnosis of pure ocular mucous membrane pemphigoid: indirect immunofluorescence versus immunoblot.
        Eur J Dermatol. 2009; 19: 456-460
        • Ong H.S.
        • Setterfield J.F.
        • Minassian D.C.
        • et al.
        Mucous membrane pemphigoid with ocular involvement: the clinical phenotype and its relationship to direct immunofluorescence findings.
        Ophthalmology. 2018; 125: 496-504
        • Grau A.E.
        • Setterfield J.
        • Saw V.P.
        How to do conjunctival and buccal biopsies to investigate cicatrising conjunctivitis: improving the diagnosis of ocular mucous membrane pemphigoid.
        Br J Ophthalmol. 2013; 97: 530-531
        • Thorne J.E.
        • Anhalt G.J.
        • Jabs D.A.
        Mucous membrane pemphigoid and pseudopemphigoid.
        Ophthalmology. 2004; 111: 45-52
        • Bernauer W.
        • Elder M.J.
        • Leonard J.N.
        • et al.
        The value of biopsies in the evaluation of chronic progressive conjunctival cicatrisation.
        Graefes Arch Clin Exp Ophthalmol. 1994; 232: 533-537
        • Leonard J.N.
        • Hobday C.M.
        • Haffenden G.P.
        • et al.
        Immunofluorescent studies in ocular cicatricial pemphigoid.
        Br J Dermatol. 1988; 118: 209-217
        • Dart J.K.
        The 2016 Bowman Lecture. Conjunctival curses: scarring conjunctivitis 30 years on.
        Eye (Lond). 2017; 31: 301-332
        • Domloge-Hultsch N.
        • Gammon W.R.
        • Briggaman R.A.
        • et al.
        Epiligrin, the major human keratinocyte integrin ligand, is a target in both an acquired autoimmune and an inherited subepidermal blistering skin disease.
        J Clin Invest. 1992; 90: 1628-1633
        • Bernard P.
        • Prost C.
        • Durepaire N.
        • et al.
        The major cicatricial pemphigoid antigen is a 180-kD protein that shows immunologic cross-reactivities with the bullous pemphigoid antigen.
        J Invest Dermatol. 1992; 99: 174-179
        • Roh J.Y.
        • Yee C.
        • Lazarova Z.
        • et al.
        The 120-kDa soluble ectodomain of type XVII collagen is recognized by autoantibodies in patients with pemphigoid and linear IgA dermatosis.
        Br J Dermatol. 2000; 143: 104-111
        • Tyagi S.
        • Bhol K.
        • Natarajan K.
        • et al.
        Ocular cicatricial pemphigoid antigen: partial sequence and biochemical characterization.
        Proc Natl Acad Sci U S A. 1996; 93: 14714-14719
        • Balding S.D.
        • Prost C.
        • Diaz L.A.
        • et al.
        Cicatricial pemphigoid autoantibodies react with multiple sites on the BP180 extracellular domain.
        J Invest Dermatol. 1996; 106: 141-146
        • Bhol K.C.
        • Goss L.
        • Kumari S.
        • et al.
        Autoantibodies to human alpha6 integrin in patients with oral pemphigoid.
        J Dental Res. 2001; 80: 1711-1715
        • Chan L.S.
        • Yancey K.B.
        • Hammerberg C.
        • et al.
        Immune-mediated subepithelial blistering diseases of mucous membranes. Pure ocular cicatricial pemphigoid is a unique clinical and immunopathological entity distinct from bullous pemphigoid and other subsets identified by antigenic specificity of autoantibodies.
        Arch Dermatol. 1993; 129: 448-455
        • Setterfield J.
        • Shirlaw P.J.
        • Kerr-Muir M.
        • et al.
        Mucous membrane pemphigoid: a dual circulating antibody response with IgG and IgA signifies a more severe and persistent disease.
        Br J Dermatol. 1998; 138: 602-610
        • Zillikens D.
        Diagnosis of autoimmune bullous skin diseases.
        Clin Lab. 2008; 54: 491-503
        • Saw V.P.
        • Dart J.K.
        Ocular mucous membrane pemphigoid: diagnosis and management strategies.
        Ocul Surf. 2008; 6: 128-142
        • Wieland C.N.
        • Comfere N.I.
        • Gibson L.E.
        • et al.
        Anti-bullous pemphigoid 180 and 230 antibodies in a sample of unaffected subjects.
        Arch Dermatol. 2010; 146: 21-25
        • Giurdanella F.
        • Nijenhuis A.M.
        • Diercks G.F.H.
        • et al.
        Keratinocyte footprint assay discriminates antilaminin-332 pemphigoid from all other forms of pemphigoid diseases.
        Br J Dermatol. 2020; 182: 373-381
        • van Beek N.
        • Rentzsch K.
        • Probst C.
        • et al.
        Serological diagnosis of autoimmune bullous skin diseases: prospective comparison of the BIOCHIP mosaic-based indirect immunofluorescence technique with the conventional multi-step single test strategy.
        Orphanet J Rare Dis. 2012; 7: 49
        • Tauber J.
        Ocular cicatricial pemphigoid.
        Ophthalmology. 2008; 115 (author reply 1640–1631): 1639-1640
        • Labowsky M.T.
        • Stinnett S.S.
        • Liss J.
        • et al.
        Clinical implications of direct immunofluorescence findings in patients with ocular mucous membrane pemphigoid.
        Am J Ophthalmol. 2017; 183: 48-55
        • Hendriks J.
        • Stals C.
        • Versteilen A.
        • et al.
        Stability studies of binding and functional anti-vaccine antibodies.
        Bioanalysis. 2014; 6: 1385-1393
        • Dard C.
        • Bailly S.
        • Drouet T.
        • et al.
        Long-term sera storage does not significantly modify the interpretation of toxoplasmosis serologies.
        J Microbiol Methods. 2017; 134: 38-45
        • Murakami H.
        • Nishioka S.
        • Setterfield J.
        • et al.
        Analysis of antigens targeted by circulating IgG and IgA autoantibodies in 50 patients with cicatricial pemphigoid.
        J Dermatol Sci. 1998; 17: 39-44
        • Leverkus M.
        • Schmidt E.
        • Lazarova Z.
        • et al.
        Antiepiligrin cicatricial pemphigoid: an underdiagnosed entity within the spectrum of scarring autoimmune subepidermal bullous diseases?.
        Arch Dermatol. 1999; 135: 1091-1098
        • Schmidt E.
        • Skrobek C.
        • Kromminga A.
        • et al.
        Cicatricial pemphigoid: IgA and IgG autoantibodies target epitopes on both intra- and extracellular domains of bullous pemphigoid antigen 180.
        Br J Dermatol. 2001; 145: 778-783
        • Setterfield J.
        • Theron J.
        • Vaughan R.W.
        • et al.
        Mucous membrane pemphigoid: HLA-DQB1∗0301 is associated with all clinical sites of involvement and may be linked to antibasement membrane IgG production.
        Br J Dermatol. 2001; 145: 406-414
        • Carrozzo M.
        • Cozzani E.
        • Broccoletti R.
        • et al.
        Analysis of antigens targeted by circulating IgG and IgA antibodies in patients with mucous membrane pemphigoid predominantly affecting the oral cavity.
        J Periodontol. 2004; 75: 1302-1308
        • Oyama N.
        • Setterfield J.F.
        • Powell A.M.
        • et al.
        Bullous pemphigoid antigen II (BP180) and its soluble extracellular domains are major autoantigens in mucous membrane pemphigoid: the pathogenic relevance to HLA class II alleles and disease severity.
        Br J Dermatol. 2006; 154: 90-98
        • Calabresi V.
        • Carrozzo M.
        • Cozzani E.
        • et al.
        Oral pemphigoid autoantibodies preferentially target BP180 ectodomain.
        Clin Immunol. 2007; 122: 207-213
        • Bernard P.
        • Antonicelli F.
        • Bedane C.
        • et al.
        Prevalence and clinical significance of anti-laminin 332 autoantibodies detected by a novel enzyme-linked immunosorbent assay in mucous membrane pemphigoid.
        JAMA Dermatol. 2013; 149: 533-540
        • Hayakawa T.
        • Furumura M.
        • Fukano H.
        • et al.
        Diagnosis of oral mucous membrane pemphigoid by means of combined serologic testing.
        Oral Surg Oral Med Oral Pathol Oral Radiol. 2014; 117: 483-496
        • Cozzani E.
        • Di Zenzo G.
        • Calabresi V.
        • et al.
        Autoantibody profile of a cohort of 78 Italian patients with mucous membrane pemphigoid: correlation between reactivity profile and clinical involvement.
        Acta Derm Venereol. 2016; 96: 768-773
        • Desai N.
        • Allen J.
        • Ali I.
        • et al.
        Autoantibodies to basement membrane proteins BP180 and BP230 are commonly detected in normal subjects by immunoblotting.
        Australas J Dermatol. 2008; 49: 137-141
        • Hachisuka H.
        • Kurose K.
        • Karashima T.
        • et al.
        Serum from normal elderly individuals contains anti-basement membrane zone antibodies.
        Arch Dermatol. 1996; 132: 1201-1205