10.5826/dpc.1202a116
Autoimmune bullous diseases (AIBDs) are a group of skin-related disorders that involve damage to structures maintaining cell-cell adhesion, such as desmosomes and hemidesmosomes. Key autoimmune bullous diseases include pemphigus related diseases, pemphigoid related conditions, acquired epidermolysis bullosa (EBA), and dermatitis herpetiformis (DH). Each group of conditions exhibits characteristic clinical lesion patterns and is associated with specific autoantibodies targeting epidermal and dermal structures involved in cell-cell adhesion and skin integrity. Pemphigus diseases primarily target desmoglein (Dsg) 3 and Dsg1 proteins but several non-desmoglein autoantibodies have also been linked to pemphigus.
Pemphigoid diseases typically target BP (bullous pemphigoid)180 and BP230; EBA is associated with antibodies directed against anti-type VII collagen; and DH by IgA autoantibodies against tissue transglutaminase and deaminated gliadin. Investigation into the serological biomarkers found in autoimmune bullous diseases have allowed the development of diagnostic assessments (ie. tissue antibody detection and serological testing) based on the unique autoantibody profiles of a particular disease group.&nbsp; The methods for the detection and quantification of disease-associated autoantibodies continue to evolve and improve.
text/xml
2160-9381
2005
en
Iswariya Venkataraman
https://creativecommons.org/licenses/by-nc/4.0
Dermatology Practical & Conceptual
antibodies
Serological Biomarkers and Their Detection in Autoimmune Bullous Skin Diseases
Text.Serial.Journal
https://dpcj.org/index.php/dpc
Review
2022-05-02
2021-09-07
2022-05-02
2022-05-24
e2022116
Dermatol Pract Concept
Autoimmune bullous diseases (AIBDs) are a group of skin-related disorders that involve damage to structures maintaining cell-cell adhesion, such as desmosomes and hemidesmosomes. Key autoimmune bullous diseases include pemphigus related diseases, pemphigoid related conditions, acquired epidermolysis bullosa (EBA), and dermatitis herpetiformis (DH). Each group of conditions exhibits characteristic clinical lesion patterns and is associated with specific autoantibodies targeting epidermal and dermal structures involved in cell-cell adhesion and skin integrity. Pemphigus diseases primarily target desmoglein (Dsg) 3 and Dsg1 proteins but several non-desmoglein autoantibodies have also been linked to pemphigus.
Pemphigoid diseases typically target BP (bullous pemphigoid)180 and BP230; EBA is associated with antibodies directed against anti-type VII collagen; and DH by IgA autoantibodies against tissue transglutaminase and deaminated gliadin. Investigation into the serological biomarkers found in autoimmune bullous diseases have allowed the development of diagnostic assessments (ie. tissue antibody detection and serological testing) based on the unique autoantibody profiles of a particular disease group.&nbsp; The methods for the detection and quantification of disease-associated autoantibodies continue to evolve and improve.
Iswariya Venkataraman
2022/05/02
10.5826/dpc.1202a116
e2022116
EUROIMMUN US Inc, 1 Bloomfield Ave, Mountain Lakes, New Jersey, United States 
2160-9381
antibodies
en
e2022116
Alaeen H, Toosi R, Mahmoudi H, et al. Short-term clinical and serological follow-up with conventional and conformational anti-desmoglein antibodies in treatment-naïve and previously treated patients with pemphigus vulgaris after receiving rituximab. Int J Womens Dermatol. 2019;5(5):372-377. DOI: 10.1016/j.ijwd.2019.05.008. PMID: 31909160. PMCID: PMC6938821.
Serological Biomarkers and Their Detection in Autoimmune Bullous Skin Diseases
https://dpcj.org/index.php/dpc/article/download/2005/1587
                    Dermatology Practical & Conceptual


           Serological Biomarkers and Their Detection
              in Autoimmune Bullous Skin Diseases
        Ilana Heckler1, Michael Hong2,3, Animesh Amart Sinha3, Iswariya Venkataraman1


1 EUROIMMUN US Inc, 1 Bloomfield Ave, Mountain Lakes, New Jersey, United States
2 College of Medicine, Florida State University, Tallahassee, Florida, United States
3 Department of Dermatology, Jacobs School of Medicine, University at Buffalo, Buffalo, New York, United States



Key words: diagnostics, pemphigoid, pemphigus, antibodies
Citation: Heckler I, Hong M, Sinha A, Venkataraman I. Serological Biomarkers and Their Detection in Autoimmune Bullous Skin Diseases,
Dermatol Pract Concept. 2022;12(2):e2022116. DOI: https://doi.org/10.5826/dpc.1202a116
Accepted: November 22, 2021; Published: April 2022
Copyright: ©2022 Heckler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-
NonCommercial License (BY-NC-4.0), https://creativecommons.org/licenses/by-nc/4.0/, which permits unrestricted noncommercial use,
distribution, and reproduction in any medium, provided the original authors and source are credited.
Funding: None.
Competing interests: None.
Authorship: All authors have contributed significantly to this publication
Corresponding author: Iswariya Venkataraman, Ph.D., EUROIMMUN US, 1 Bloomfield Ave, Mountain Lakes, New Jersey, United States.
E-mail: iswariya.venkataraman@euroimmun.us



  ABSTRACT             Autoimmune bullous diseases (AIBDs) are a group of skin-related disorders that involve damage
                       to structures maintaining cell-cell adhesion, such as desmosomes and hemidesmosomes. Key AIBDs
                       include pemphigus related diseases, pemphigoid related conditions, acquired epidermolysis bullosa
                       (EBA), and dermatitis herpetiformis (DH). Each group of conditions exhibits characteristic clinical
                       lesion patterns and is associated with specific autoantibodies targeting epidermal and dermal struc-
                       tures involved in cell-cell adhesion and skin integrity. Pemphigus diseases primarily target desmoglein
                       (Dsg) 3 and Dsg1 proteins but several non-Dsg autoantibodies have also been linked to pemphigus.
                           Pemphigoid diseases typically target bullous pemphigoid (BP)180 and BP230; EBA is associated
                       with antibodies directed against anti-type VII collagen and DH by IgA autoantibodies against tis-
                       sue transglutaminase and deaminated gliadin. Investigation into the serological biomarkers found in
                       AIBDs have allowed the development of diagnostic assessments (i.e. tissue antibody detection and
                       serological testing) based on the unique autoantibody profiles of a particular disease group. The meth-
                       ods for the detection and quantification of disease-associated autoantibodies continue to evolve and
                       improve.



Introduction                                                            protective barrier, regulation of epidermal temperature and
                                                                        nutrients and other functions of the epidermis are dependent
The skin is the largest organ of the body, protecting us from           upon the maintenance of stable connections between kerat-
the environment, regulating body temperature and permit-                inocytes and other epidermal structures primarily mediated
ting the sensation of touch. It is divided into 3 main lay-             by adhesive desmosomal and hemidesmosomal proteins [2].
ers: the outermost epidermis layer, the middle dermis layer                  Desmosomes are specialized tight junctions critical to
and the lower hypodermis layer [1]. The epidermis is mainly             cellular adhesion (Figure 1). They are arranged on adja-
made up of keratin producing cells called keratinocytes. The            cent sides of plasma membranes and can be seen in tissues



Review | Dermatol Pract Concept. 2022;12(2):e2022116                                                                                1
       Figure 1. Graphical representation of human skin. Desmosomes connect adjacent keratinocytes and are critical to cellular
       adhesion (top circle within desmosomes: there is a vast network of cadherin proteins [desmogleins, desmocollins and des-
       moplakins]), linker proteins [eg plakoglobin, plakophilin] and keratin intermediate filaments). Hemidesmosomes facilitate
       the adhesion of basal keratinocytes to the underlying basal lamina. Key hemidesmosomal-associated proteins include the
       cytoplasmic protein BP230, the transmembrane protein BP180, laminin 332 and collagen type VII.


including cardiac muscle, gastrointestinal mucosa, and epi-          BP180, laminin 332 and collagen type VII [5]. Another fea-
thelia, all of which can be subject to significant mechanical        ture of hemidesmosomes is their role in signaling pathways,
stress during normal physiological and disease states [3].           relevant for the migration of keratinocytes (Figure 1) [6].
    Within desmosomes, there is a vast network of cadherin               Autoimmunity involves the presence of antibodies (pro-
proteins (desmogleins, desmocollins and desmoplakins),               duced by B lymphocytes) and T lymphocytes that have
linker proteins (eg plakoglobin, plakophilin) and keratin            escaped mechanisms of self-tolerance, both centrally and
                                                                     ­
intermediate filaments, that connect as desmosome-inter-             peripherally, that are reactive to one’s own self-antigens.
                                                                     ­
mediate filament complexes (DIFCs). Desmoplakin, which               When auto-reactive lymphocytes cause enough target tissue
coordinates other cadherin proteins and keratin filaments, is        damage, autoimmune disease can occur.
the most prevalent protein within the desmosome [4].                     There are over 80 human autoimmune diseases in ex-
    Hemidesmosomes resemble tiny stud-shaped structures              istence, affecting over 20 million Americans [7]. Indicators
and are similar in shape to desmosomes. However, there are           of autoimmunity (ie antinuclear antibodies) suggest that the
several differences between these 2 structural components.           incidence of autoimmune disease has been increasing over
Hemidesmosomes attach keratinocytes to the extracellular             the past few decades [8].
matrix and utilize integrins rather than desmogleins and des-            Collectively, autoimmune diseases present a tremendous,
mocollins. Key hemidesmosomal-associated proteins include            and likely under-estimated burden on healthcare costs: over
the cytoplasmic protein BP230, the transmembrane protein             $100 billion annually in the United States [9]. These costs



2                                                                                Review | Dermatol Pract Concept. 2022;12(2):e2022116
reflect a multitude of factors which are impacted by delays in        Objectives
diagnosis, poor or infrequent monitoring of disease activity
leading to less than optimal disease management.                      Here, we discuss the key autoantigens in AIBDs and high-
      Systemic autoimmune diseases affect multiple organs,            light how serological testing can be used in conjunction with
whereas organ-specific diseases target a single organ such as         clinical symptoms for diagnostic purposes.
the skin. Autoimmune diseases that affect the skin include
vitiligo, scleroderma, lupus, psoriasis, vasculitis, and autoim-
                                                                      Pemphigus diseases
mune bullous dermatoses (AIBDs). AIBDs are a collection of
autoimmune skin specific disease characterized by the pro-            Pemphigus diseases are commonly characterized by the pro-
duction of autoantibodies against structural components of            duction of autoantibodies primarily against the desmosomal
the skin including desmosomes and hemidesmosomes [10].                proteins desmoglein (Dsg)3 and Dsg1 which results in the
Such an autoimmune reaction interferes with intercellular             loss of epidermal cell-cell adhesion and subsequent blister
connections within the epidermis in addition to the crucial           formation [11]. Some patients experience only mucous mem-
linkage between the epidermis and the dermis. AIBDs man-              brane erosions with minimal skin blistering, others exhibit
ifest as skin layer separation and blistering and are divided         lesions on both mucosal as well as non-mucosal surfaces,
into 4 main groups according to their target antigens and             while others still may only show skin involvement without
localization of the blisters: pemphigus diseases, pemphigoid          mucous membrane involvement [11]. The clinical phenotype
diseases, acquired epidermolysis bullosa (EBA), and dermati-          of pemphigus has been linked to defined Dsg3 and Dsg1
tis herpetiformis (DH) (Table 1) [10].                                antibody profiles [12]. Additionally, differences in the nor-
      Clearly, there is need for a greater understanding of the       mal tissue distribution of Dsg1 and Dsg3 proteins (Dsg1 on
epidemiology, pathophysiology, and natural history of AIBD.           the epidermal surface and Dsg3 in deep epidermal layers/
The continued evolution of methods for the reliable, acces-           mucous membranes) may explain the varying clinical man-
sible, and cost-effective detection of disease relevant auto-         ifestations of different pemphigus forms [13]. For example,
antibodies is an ongoing endeavor to improve our ability to           in pemphigus foliaceus (PF), IgG antibodies are only directed
diagnose and monitor autoimmune activity, with impact in              against Dsg1 and blistering is confined to the skin surface.
clinical management and decision-making accurately and                On the other hand, in pemphigus vulgaris (PV), autoanti-
rapidly.                                                              bodies against both Dsg1 and Dsg3 can be observed, and



                             Table 1. Target antigens in autoimmune bullous dermatoses
            AIBD Subtype                       Blister Location               Target Antigena                       Ig Type
    Pemphigus
    PV (mucosal-dominant type)           Intraepidermal            Dsg3                                       IgG
    PV (mucocutaneous type)              Intraepidermal            Dsg3, Dsg1, Dsc1, Dsc2, Dsc3               IgG
    IgA Pemphigus                        Intraepidermal            Dsg3, Dsg1, desmocollins
    PF                                   Intraepidermal            Dsg1                                       IgG
    PNP                                  Intraepidermal            Envoplakin, Dsg3, Dsg1, periplakin,        IgG
                                                                   epiplakin, plectin, desmoplakins, Dsc(1-
                                                                   3), BP230, α2-macroglobulin-like 1
    Pemphigoid
    BP                                   Subepidermal              BP180, BP230                               IgG
    MMP                                  Subepidermal              BP180, BP230, laminin332, integrin α6/     IgG
                                                                   β4, and collagen VII
    EBA                                  Subepidermal              Type VII collagen                          IgG
    DH                                   Subepidermal              Epidermal/tissue transglutaminase,         IgA/IgG
                                                                   endomysium, deamidated gliadin
    Pemphigoid gestationis               Subepidermal              BP180, BP230                               IgG
    Linear IgA bullous dermatosis        Subepidermal              Ectodomain fragment of BP180, BP230        IgA
a
 Main target antigens are indicated in bold.

AIBD = autoimmune bullous dermatoses; PV = pemphigus vulgaris; PF = pemphigus foliaceus; Dsc = desmocollins; PNP = paraneoplastic
pemphigus; BP = bullous pemphigoid; MMP = mucous membrane pemphigoid; EBA = epidermolysis bullosa acquisita; DH = dermatitis
herpetiformis; Dsg = desmoglein.



Review | Dermatol Pract Concept. 2022;12(2):e2022116                                                                             3
the degree of blistering and mucous membrane involvement            diseases occur when the immune system produces autoanti-
varies based on the prevalence of either anti-Dsg1 and an-          bodies against proteins involved in the linkage between the
ti-Dsg3 [12]. This framework, correlating the clinical presen-      epidermis and dermis. As a result of this autoimmune reac-
tation of pemphigus to antibody profile, is known as the Dsg        tion, the epidermal layer separates from the dermis. Several
compensation hypothesis (DCH), featured prominently in              different types of pemphigoid diseases exist including bullous
dermatology textbooks and previous research studies [14].           pemphigoid (BP), pemphigoid gestationis, mucous membrane
    As elegant as the hypothesis may be, however, recent            pemphigoid, linear IgA dermatosis and p200 pemphigoid [10].
studies have identified exceptions to this hypothesis [15,16].         The hemidesmosomal proteins, BP180 and BP230, which
    PV accounts for 80% of all pemphigus cases and mainly           tether the 2 skin layers together, are the common autoanti-
affects middle-aged and elderly populations [17]. While Dsg3        body targets in BP. BP is the most common AIBD in the gen-
(89% - 90% of patients) and Dsg1 (50% - 60% of patients)            eral population, with an annual incidence ranging between 2.3
are the major autoantigens in PV, additional structural and         to 23 cases per million. BP disproportionately affects elderly
metabolic autoantigens have been identified including desmo-        people, with an incidence of 190-312 cases per million among
collins (Dsc) 1 and 3, muscarinic and nicotinic acetylcholine re-   those 80 years and older [14]. This disease manifests with
ceptors, mitochondrial antigens, thyroid peroxidase, hSPCA1,        bulging skin blisters and minimal mucous membrane involve-
plakophilin 3, plakoglobin, and E-cadherin [18]. Studies have       ment [25]. Unlike pemphigus, BP shows a negative Nikolsky
shown that autoantibodies against these additional targets may      sign (ie no splitting of skin upon applying pressure) [26].
complement the effects of anti-Dsg autoantibodies and explain          Autoantibodies against BP180 represent the most sig-
individual variations in pemphigus disease severity [18].           nificant biomarker in BP due to their high prevalence [27].
    In paraneoplastic pemphigus (PNP), autoantibodies are           Additional screening for anti-BP230 antibodies is important
directed against desmosomes including Dsg1 and Dsg3,                as they occur in 40% of patients who are seronegative for
α2-macroglobulin-like 1, and the plakins envoplakin, desmo-         anti-BP180 antibodies. The parallel detection of both an-
plakin I and II, plectin, periplakin and the hemidesmosome          ti-BP230 antibodies and anti-BP180 antibodies increases
BP230. The presence of desmoplakin autoantibodies is also           the sensitivity of BP detection significantly, to a combined
common to PV, PF, and BP. However, autoantibodies for envo-         97.1% [28]. Pemphigoid gestationis is the manifestation of
plakin and periplakin on immunoblot, as well as autoanti-           BP in pregnant women and is characterized by autoantibod-
bodies for desmoplakin (on indirect immunofluorescence and          ies predominately against epitopes in the immunodominant
rat bladder epithelium), appear to be sensitive and specific        NC16A domain of BP180 (BP180-NC16A) [29]. In chil-
for PNP diagnosis [19]. This has led to the development of          dren, linear IgA dermatosis occurs from the autoantibody
an enzyme-linked immunosorbent assay (ELISA) that detects           recognition of the ectodomain fragment of BP180 [30]. In
envoplakin in consideration for a diagnostic tool for PNP.          addition to BP180, laminin 332 is a major target in mucous
    PNP is associated in a majority of cases with non-Hod-          membrane pemphigoid (MMP) [31]. Additionally, patients
gkin lymphoma, chronic lymphocytic leukemia and Castle-             with mucous membrane pemphigoid may show antibodies
man disease [20]. A common clinical feature is stomatitis           against BP230, integrin α6/β4, and collagen VII [31,32]. The
which presents with painful erosions and ulcerations of the         identification of anti-laminin 332 is important for determin-
oropharynx. Anti-envoplakin antibodies are highly specific          ing a patients prognosis as anti-laminin 332 positive patients
for PNP and are used for the differentiation of PNP from            seem to be at an increased risk of malignancies [33].
other AIBDs [21].
    In IgA pemphigus, a rare form of pemphigus with unclear
                                                                    Epidermolysis bullosa acquisita
etiology, serum IgA autoantibodies are associated with reac-
tivity against the desmosomal cadherins Dsc1, Dsc2, Dsc3,           EBA is a severe blistering dermatosis characterized by autoanti-
Dsg1, and Dsg3 [17]. These circulating IgA antibodies lead          bodies against type VII collagen [34]. EBA manifests as subepi-
to formation of pruritic and painful eruptions that present         dermal blisters and erosions in response to the minor irritation
as vesicles and pustules on the skin [22]. As IgA pemphigus         of skin and affects both the skin and mucous membranes. The
is so rare, there is currently no reported sex, age, or race dis-   level of the cleavage in the basal membrane contributes to the
tribution of this disease. However, IgA pemphigus has been          various phenotypes of EBA, including the most common inflam-
observed in all age demographics [23].                              matory and mechanobullous (noninflammatory) variants [35].


Pemphigoid diseases                                                 Dermatitis herpetiformis
Pemphigoid diseases are characterized by subepidermal blister       DH is an itchy dermatosis affecting 10% of celiac patients. It
formation in the skin and mucous membranes [24]. Pemphigoid         manifests as blisters in the subepidermis of areas such as the



4                                                                              Review | Dermatol Pract Concept. 2022;12(2):e2022116
elbows, knees, and buttocks. There is also minimal blistering      autoantibodies against Dsg1 and Dsg3, with a sensitivity of
of the mucous membranes. DH is one of many manifestations          81% - 100% and a specificity of 89% - 100% [17,41]. For
of gluten-sensitivity and is characterized by IgA autoantibod-     the differentiation of autoantibodies in subepidermal AIBDs,
ies against endomysium tissue/epidermal transglutaminase           tissue sections of salt-split skin are used [42]. Salt-split skin
(anti-tTG/-eTG) and/or deamidated gliadin (IgA/IgG) [36].          substrate has a sensitivity of 73% - 96% and a specificity
In contrast to the increase in diagnosis of celiac disease, DH     of 97% for such subepidermal antibodies. Additionally, as
incidence appears to be decreasing (Table 1) [37].                 antibodies have varying antigenic binding properties on
                                                                   salt-split skin, this allows for the differentiation between the
                                                                   subepidermal AIBDs BP, pemphigoid gestationis, linear IgA
Diagnostic approach                                                dermatosis and other subepidermal AIBDs such as EBA, and
The diagnosis of AIBDs requires the detection of both cir-         anti-laminin-332-type MMP. Where BP180 and BP230 are
culating and tissue-bound antibodies, and histopathology,          located on the epidermal side of salt-split skin, collagen type
in conjunction with clinical symptoms [38]. The pathway            VII and laminin 332 remain on the dermal side.
to AIBD diagnosis can be broken down into 4 pillars. First,           Urinary bladder is an ideal substrate for distinguishing
the clinical manifestations of the disease must be assessed.       between PNP and other pemphigus diseases as plakins like
Second, histopathology can be performed to provide infor-          envoplakin are highly expressed in the bladder while Dsg1
mation on the location of skin involvement (sub- or intraepi-      and Dgs3 are not [43]. Urinary bladder is therefore a highly
dermal separation). Third, the detection of tissue bound           specific substrate for PNP (99% - 100%) and having a sensi-
autoantibodies by direct immunofluorescence (DIF) is done.         tivity of 74% [17]. Finally, liver tissue is useful for the detec-
DIF is the current diagnostic gold standard for AIBDs but          tion of IgA autoantibodies against endomysium in DH [44].
gives limited information on the target antigens. DIF has a           The International Bullous Diseases Consensus Group
sensitivity of 82% - 91% and a specificity of 98% [17]. The        recommends using IIF microscopy on monkey esophagus
fourth pillar is the identification of autoantibodies by sero-     or human skin to detect autoantibodies against surface pro-
logical testing such as indirect immunofluorescence (IIF) mi-      teins of epidermal keratinocytes [39]. In cases of atypical
croscopy, monospecific ELISA or immunoblot techniques.       17    presentation or the suspicion of another AIBD, the use of
   Serological testing for the detection of circulating an-        IIF microscopy on rat bladder and immunoblot/immuno-
tibodies in AIBDs has the advantage of being minimally             precipitation is discussed. They also describe the use of re-
invasive and may be suitable for diagnostic purposes in            combinantly expressed Dsg1, Dsg3, or envoplakin substrates
conjunction with the clinical manifestations, and for aiding       (EUROIMMUN) when Dsg- or envoplakin-specific ELISA
therapy decisions and disease prognosis [17]. Conventional         cannot be used [39].
serological detection of AIBD-specific antibodies involves an
initial IIF screen using tissue substrates, followed by an anti-   Antigen-specific detection of AIBDs
gen-specific assay such as ELISA.                                  The detection of antigen-specific autoantibodies in AIBDs
   In 2016, the International Bullous Diseases Consensus           can be achieved using monospecific IIF and ELISAs [17].
Group met to standardize the diagnosis and management              Monospecific IIF can be accomplished using transfected
of pemphigus [39]. The diagnosis of pemphigus was agreed           cells as a substrate in which the target antigen has been re-
to be based on the clinical presentation and histopathology        combinantly expressed. Additionally, designer antigens have
consistent with pemphigus and either a positive DIF micros-        been created to enhance diagnostic sensitivity and specificity
copy or serologic detection of autoantibodies against epithe-      of IIF. Such purified recombinant designer antigens are uti-
lial cell surface antigens [39]. The determination of serum        lized as monospecific IIF substrates. BIOCHIPS, which are
autoantibodies was recommended for therapeutic decision            coated with an IIF substrate and arranged onto microscope
making as serum levels of IgG against Dsg1 and Dsg3 cor-           slides, allow for autoantibody screening and confirmatory
relate with the clinical activity of pemphigus.                    discrimination in a single incubation. In this way, various
                                                                   types of AIBDs can be screened for in one test. IIF BIO-
AIBD autoantibody screening using tissue IIF                       CHIP mosaics contain combinations of different substrates
Due to their high sensitivity, tissue substrates are ideal for     (esophagus, salt-split skin, bladder mucosa, transfected cells,
screening for AIBDs autoantibodies (esophagus, salt-split          purified designer antigens). A study which compared the
skin, bladder mucosa) [40]. The esophagus substrate yields         performance of the “Dermatology Mosaic 7” with a multi-
characteristic honeycomb-like immunofluorescence pat-              step serum analysis using single antibody tests, found a 94%
terns which can be differentiated when screening for anti-         diagnostic agreement between both methods. Therefore,
bodies in suspected cases of PV or PF. IIF using esophagus         multiparametric BIOCHIP mosaics offer a cost and time ef-
as a substrate has proven to be useful for the detection of        fective IIF method.



Review | Dermatol Pract Concept. 2022;12(2):e2022116                                                                               5
    Autoantibodies can be mono-specifically identified us-          differential diagnostic clarification [46]. The detection of an-
ing ELISAs which utilize purified recombinant proteins              ti-collagen type VII antibodies aids in the detection of EBA
[17]. Commercial assays which utilize the recombinant               and allows for the differentiation of EBA from other AIBDs
ectodomains of Dsg1 and Dsg3 have a high sensitivity and            [47,48]. In addition to disease monitoring, correlations
specificity for the detection of pemphigus foliaceus and            ­between lowered levels of AIBDs specific autoantibodies in
pemphigus vulgaris (96% - 100%, 96% - 100% and 85% -                response to therapy point to the use of serological testing for
100%, 96 - 100%, respectively) [17]. In addition to their use       therapy monitoring purposes [49,50].
as monospecific substrates in IIF, designer antigens have been            Continued efforts to develop and deploy increasingly
developed for ELISAs (for the detection of antibodies against       accurate and multi-parameter methods for the detection of
BP180 and BP230, deamidated gliadin peptides) to improve            the comprehensive set of AIBD-associated autoantibodies
immunoreactivity. ELISA techniques provide quantitative             can be expected to enhance diagnostic efforts and further
measurement which is useful for the application of therapy          our understanding of disease mechanisms, progression and
monitoring. Profile ELISAs containing a combination of an-          response to therapy.
tigens enables the simultaneous detection of multiple AIBDs
subtypes in patients with suspected AIBDs.
                                                                    References
    Experts recommend determining anti-Dsg1 and/or an-
ti-Dsg3 IgG antibodies by ELISA for the detection of PF,             1. Yousef H, Alhajj M, Sharma S. Anatomy, Skin (Integument), Epi-
and mucosal/mucocutaneous PV (Mannose-Binding Lectin,                   dermis. In: StatPearls. Treasure Island (FL): StatPearls Publish-
                                                                        ing; 2021.
EUROIMMUN) [39]. Serum concentrations of antibodies
                                                                     2. Schmidt A, Koch PJ. Desmosomes: just cell adhesion or is
against Dsg1 and Dsg3 are associated with pemphigus dis-
                                                                          there more? Cell Adh Migr. 2007;1(1):28-32. DOI: 10.4161/
ease activity in and high levels of anti-Dsg1 by ELISA has                cam.1.1.4204. Epub 2007 Jan 26. PMID: 19262094. PMCID:
a positive predictive value for skin relapses. Therefore, the             PMC2633677.
determination of serum autoantibodies against skin struc-            3.   Delva E, Tucker DK, Kowalczyk AP. The desmosome. Cold Spring
tural proteins by ELISA has a prognostic value for guiding                Harb Perspect Biol. 2009;1(2):a002543. DOI: 10.1101/cshper-
                                                                          spect.a002543. PMID: 20066089. PMCID: PMC2742091.
pemphigus treatment.
                                                                     4.   Garrod D, Chidgey M. Desmosome structure, composition and
                                                                          function. Biochim Biophys Acta. 2008;1778(3):572-87. DOI:
Conclusions                                                               10.1016/j.bbamem.2007.07.014. PMID: 17854763.
                                                                     5.   Borradori L, Sonnenberg A. Structure and Function of
A definitive diagnosis of AIBD is based on a combination of               Hemidesmosomes: More Than Simple Adhesion Complexes. J
                                                                          Invest Dermatol. 1999;112(4):411-418. DOI: 10.1046/j.1523-
clinical signs and symptoms and the analysis of autoantibod-
                                                                          1747.1999.00546.x. PMID: 10201522.
ies using IIF and ELISA. IIF, using various tissue substrates, is
                                                                     6.   Harrison OJ, Brasch J, Lasso G, et al. Structural basis of adhesive
a useful application for antibody screening while transfected             binding by desmocollins and desmogleins. Proc Natl Acad Sci U S
cells and purified antigen substrates are suitable for anti-              A. 2016;113(26):7160-7165. DOI: 10.1073/pnas.1606272113.
gen-specific IIF. ELISA allows for the quantitative measure-              PMID: 27298358. PMCID: PMC4932976.
ment of antibody levels to support the detection of different        7.   Rose NR. Autoimmune Diseases. In: Quah SR, ed. International
                                                                          Encyclopedia of Public Health (Second Edition). Oxford: Aca-
AIBD subtypes. Serological antibody testing is important for
                                                                          demic Press; 2017:192-195.
distinguishing between the various AIBD subtypes due to
                                                                     8.   Dinse G, Parks C, Weinberg C, et al. Increasing Prevalence of
differences in their prognosis and treatment.                             Antinuclear Antibodies in the United States. Arthritis Rheuma-
    Immunologic testing has also a key role in providing an               tol. 2020;72(6):1026-1035. DOI: 10.1002/art.41214. PMID:
accurate diagnosis as blistering skin diseases are easily misdi-          32266792. PMCID: PMC7255943.
agnosed. Oral blisters are often misdiagnosed as an infection        9.   Julian MK. Autoimmune disease: Cost-effective care. Nurs
                                                                          Manage. 2014;45(11):24-29; quiz 30. DOI: 10.1097/01.NU-
such as candidiasis or herpes. Without a proper diagnosis,
                                                                          MA.0000455740.32485.9c. PMID: 25304753.
a patient is at risk of being mistreated, potentially with a
                                                                    10.   Egami S, Yamagami J, Amagai M. Autoimmune bullous skin
chronic overexposure to steroids which may reduce some                    diseases, pemphigus and pemphigoid. J Allergy Clin Immunol.
symptoms without fully addressing the underlying problem.                 2020;145(4):1031-1047. DOI: 10.1016/j.jaci.2020.02.013.
    Additionally, serological testing allows for the monitor-             PMID: 32272980.
ing of AIBD disease. Serum levels of anti-BP180 antibodies          11.   Kasperkiewicz M, Ellebrecht CT, Takahashi H, et al. Pem-
                                                                          phigus. Nat Rev Dis Primers. 2017;3:17026. DOI: 10.1038/
correlate with disease activity of BP while anti-BP230 levels
                                                                          nrdp.2017.26. PMID: 28492232. PMCID: PMC5901732.
correlate with the disease duration [45]. Moreover, levels of
                                                                    12.   Amagai M, Tsunoda K, Zillikens D, Nagai T, Nishikawa T. The
anti-Dsg1 and Dsg3 are associated with severity of pemphi-                clinical phenotype of pemphigus is defined by the anti-desmoglein
gus diseases and response to therapy while anti-envoplakin                autoantibody profile. J Am Acad Dermatol. 1999;40(2 Pt 1):167-
titers correlate with the degree of PNP symptoms as well as               170. DOI: 10.1016/s0190-9622(99)70183-0. PMID: 10025740.



6                                                                                 Review | Dermatol Pract Concept. 2022;12(2):e2022116
13. Amagai M, Koch PJ, Nishikawa T, Stanley JR. Pemphigus vul-           28. Yoshida M, Hamada T, Amagai M, et al. Enzyme-linked immu-
    garis antigen (desmoglein 3) is localized in the lower epider-           nosorbent assay using bacterial recombinant proteins of human
    mis, the site of blister formation in patients. J Invest Dermatol.       BP230 as a diagnostic tool for bullous pemphigoid. J Dermatol
    1996;106(2):351-355. DOI: 10.1111/1523-1747.ep12343081.                  Sci. 2006;41(1):21-30. DOI: 10.1016/j.jdermsci.2005.11.002.
    PMID: 8601740.                                                           PMID: 16364599.
14. Mahoney MG, Wang Z, Rothenberger K, Koch PJ, Amagai M,               29. Shimanovich I, Skrobek C, Rose C, et al. Pemphigoid gestatio-
    Stanley JR. Explanations for the clinical and microscopic local-         nis with predominant involvement of oral mucous membranes
    ization of lesions in pemphigus foliaceus and vulgaris. J Clin           and IgA autoantibodies targeting the C-terminus of BP180.
    Invest. 1999;103(4):461-468. DOI: 10.1172/JCI5252. PMID:                 J Am Acad Dermatol. 2002;47(5):780-784. DOI: 10.1067/
    10021453. PMCID: PMC408100.                                              mjd.2002.113677. PMID: 12399776.
15. Sinha AA, Sajda T. The Evolving Story of Autoantibodies in Pem-      30. Schumann H, Baetge J, Tasanen K, et al. The shed ectodomain of
    phigus Vulgaris: Development of the “Super Compensation Hy-              collagen XVII/BP180 is targeted by autoantibodies in different
    pothesis”. Front Med (Lausanne). 2018;5:218. DOI: 10.3389/               blistering skin diseases. Am J Pathol. 2000;156(2):685-695. DOI:
    fmed.2018.00218. PMID: 30155465. PMCID: PMC6102394.                      10.1016/S0002-9440(10)64772-4. PMID: 10666397. PMCID:
16. Ahmed AR, Carrozzo M, Caux F, et al. Monopathogenic vs mul-              PMC1850053.
    tipathogenic explanations of pemphigus pathophysiology. Exp          31. Chiorean R, Danescu S, Virtic O, et al. Molecular diagnosis of
    Dermatol. 2016;25(11):839-846. DOI: 10.1111/exd.13106.                   anti-laminin 332 (epiligrin) mucous membrane pemphigoid. Or-
    PMID: 27305362.                                                          phanet J Rare Dis. 2018;13(1):111. DOI: 10.1186/s13023-018-
17. Saschenbrecker S, Karl I, Komorowski L, et al. Serological Di-           0855-x. PMID: 29980216. PMCID: PMC6035451.
    agnosis of Autoimmune Bullous Skin Diseases. Front Immu-             32. Kamaguchi M, Iwata H. The Diagnosis and Blistering Mech-
    nol. 2019;10:1974. DOI: 10.3389/fimmu.2019.01974. PMID:                  anisms of Mucous Membrane Pemphigoid. Front Immu-
    31552014. PMCID: PMC6736620.                                             nol. 2019;10:34. DOI: 10.3389/fimmu.2019.00034. PMID:
18. Amber KT, Valdebran M, Grando SA. Non-Desmoglein An-                     30740099. PMCID: PMC6357922.
      tibodies in Patients With Pemphigus Vulgaris. Front Immu-          33. Egan CA, Lazarova Z, Darling TN, Yee C, Coté T, Yancey KB.
      nol. 2018;9:1190. DOI: 10.3389/fimmu.2018.01190. PMID:                 Anti-epiligrin cicatricial pemphigoid and relative risk for can-
      29915578. PMCID: PMC5994403.                                           cer. Lancet. 2001;357(9271):1850-1851. DOI: 10.1016/S0140-
19.   Powell JG, Grover RK, Plunkett RW, Seiffert-Sinha K, Sinha AA.         6736(00)04971-0. PMID: 11410196.
      Evaluation of a Newly Available ELISA for Envoplakin Autoan-       34. Mehren CR, Gniadecki R. Epidermolysis bullosa acquisita: cur-
      tibodies for the Diagnosis of Paraneoplastic Pemphigus. J Drugs        rent diagnosis and therapy. Dermatol Reports. 2011;3(3):e38.
      Dermatol. 2015;14(10):1103-1106. PMID: 26461820.                       DOI: 10.4081/dr.2011.e38. PMID: 25386290. PMCID:
20.   Anhalt GJ. Paraneoplastic pemphigus. J Investig Derma-                 PMC4211502.
      tol Symp Proc. 2004;9(1):29-33. DOI: 10.1111/j.1087-               35. Kridin K, Kneiber D, Kowalski EH, Valdebran M, Amber KT.
      0024.2004.00832.x. PMID: 14870982.                                     Epidermolysis bullosa acquisita: A comprehensive review. Au-
21.   Poot AM, Diercks GF, Kramer D, et al. Laboratory diagnosis of          toimmunity Reviews. 2019;18(8):786-795. DOI: 10.1016/j.au-
      paraneoplastic pemphigus. Br J Dermatol. 2013;169(5):1016-             trev.2019.06.007. PMID: 31181325.
      1024. DOI: 10.1111/bjd.12479. PMID: 23796242.                      36. Antiga E, Caproni M. The diagnosis and treatment of dermati-
22.   Hashimoto T, Teye K, Hashimoto K, et al. Clinical and Immu-            tis herpetiformis. Clin Cosmet Investig Dermatol. 2015;8:257-
      nological Study of 30 Cases With Both IgG and IgA Anti-Kera-           265. DOI: 10.2147/CCID.S69127. PMID: 25999753. PMCID:
      tinocyte Cell Surface Autoantibodies Toward the Definition of          PMC4435051.
      Intercellular IgG/IgA Dermatosis. Front Immunol. 2018;9:994.       37. Salmi TT. Dermatitis herpetiformis. Clin Exp Derma-
      DOI: 10.3389/fimmu.2018.00994. PMID: 29867971. PMCID:                  tol. 2019;44(7):728-731. DOI: 10.1111/ced.13992. PMID:
      PMC5950707.                                                            31093998.
23.   Kridin K. Pemphigus group: overview, epidemiology, mortality,      38. Witte M, Zillikens D, Schmidt E. Diagnosis of Autoimmune
      and comorbidities. Immunol Res. 2018;66(2):255-270. DOI:               Blistering Diseases. Front Med (Lausanne). 2018;5:296. DOI:
      10.1007/s12026-018-8986-7. PMID: 29479654.                             10.3389/fmed.2018.00296. PMID: 30450358. PMCID:
24.   Miyamoto D, Santi CG, Aoki V, Maruta CW. Bullous pemphi-               PMC6224342.
      goid. An Bras Dermatol. 2019;94(2):133-146. DOI: 10.1590/          39. Murrell DF, Peña S, Joly P, et al. Diagnosis and management
      abd1806-4841.20199007.         PMID:     31090818.     PMCID:          of pemphigus: Recommendations of an international panel of
      PMC6486083.                                                            experts. J Am Acad Dermatol. 2020;82(3):575-585.e1. DOI:
25.   Kridin K, Ludwig RJ. The Growing Incidence of Bullous Pem-             10.1016/j.jaad.2018.02.021. PMID: 29438767. PMCID:
      phigoid: Overview and Potential Explanations. Front Med (Lau-          PMC7313440.
      sanne). 2018;5:220. DOI: 10.3389/fmed.2018.00220. PMID:            40. Shetty VM, Subramaniam K, Rao R. Utility of immunofluores-
      30177969. PMCID: PMC6109638.                                           cence in dermatology. Indian Dermatol Online J. 2017;8(1):1-8.
26.   Kershenovich R, Hodak E, Mimouni D. Diagnosis and classifi-            DOI: 10.4103/2229-5178.198774. PMID: 28217464. PMCID:
      cation of pemphigus and bullous pemphigoid. Autoimmun Rev.             PMC5297263.
      2014;13(4-5):477-481. DOI: 10.1016/j.autrev.2014.01.011.           41. Feibelman C, Stolzner G, Provost TT. Pemphigus Vulgaris: Su-
      PMID: 24424192.                                                        perior Sensitivity of Monkey Esophagus in the Determination
27.   Ishiura N, Fujimoto M, Watanabe R, et al. Serum levels of IgE          of Pemphigus Antibody. Arch Dermatol. 1981;117(9):561-562.
      anti-BP180 and anti-BP230 autoantibodies in patients with bul-         DOI: 10.1001/archderm.117.9.561. PMID: 7027966.
      lous pemphigoid. J Dermatol Sci. 2008;49(2):153-161. DOI:          42. Ghohestani RF, Nicolas JF, Rousselle P, Claudy AL. Diagnostic
      10.1016/j.jdermsci.2007.08.008. PMID: 17920818.                        value of indirect immunofluorescence on sodium chloride-split



Review | Dermatol Pract Concept. 2022;12(2):e2022116                                                                                       7
      skin in differential diagnosis of subepidermal autoimmune                     Dent. 2020;12(5):e440-e445. DOI: 10.4317/jced.56289. PMID:
      bullous dermatoses. Arch Dermatol. 1997;133(9):1102-1107.                     32509225. PMCID: PMC7263783.
      PMID: 9301587.                                                          47.   Koga H, Teye K, Yamashita K, Ishii N, Tsuruta D, Nakama T. De-
43.   Liu AY, Valenzuela R, Helm TN, Camisa C, Melton AL, Berg-                     tection of anti-type VII collagen IgE antibodies in epidermolysis
      feld WF. Indirect immunofluorescence on rat bladder transitional              bullosa acquisita. Br J Dermatol. 2019;180(5):1107-1113. DOI:
      epithelium: a test with high specificity for paraneoplastic pem-              10.1111/bjd.17310. PMID: 30311191.
      phigus. J Am Acad Dermatol. 1993;28(5 Pt 1):696-699. DOI:               48.   Kim JH, Kim YH, Kim S, et al. Serum levels of anti-type VII
      10.1016/0190-9622(93)70095-b. PMID: 7684408.                                  collagen antibodies detected by enzyme-linked immunosor-
44.   Wolf J, Jahnke A, Fechner K, et al. Primate liver tissue as an alter-         bent assay in patients with epidermolysis bullosa acquisita are
      native substrate for endomysium antibody immunofluorescence                   correlated with the severity of skin lesions. J Eur Acad Der-
      testing in diagnostics of paediatric coeliac disease. Clin Chim               matol Venereol. 2013;27(2):e224-e230. DOI: 10.1111/j.1468-
      Acta. 2016;460:72-77. DOI: 10.1016/j.cca.2016.06.023. PMID:                   3083.2012.04617.x. PMID: 22731917.
      27346479.                                                               49.   Mutasim DF. Therapy of autoimmune bullous diseases. Ther Clin
45.   Chanprapaph K, Ounsakul V, Pruettivorawongse D, Thadanipon                    Risk Manag. 2007;3(1):29-40. DOI: 10.2147/tcrm.2007.3.1.29.
      K. Anti-BP180 and anti-BP230 enzyme-linked immunosorbent                      PMID: 18360613. PMCID: PMC1936286.
      assays for diagnosis and disease activity tracking of bullous           50.   Alaeen H, Toosi R, Mahmoudi H, et al. Short-term clinical and
      pemphigoid: A prospective cohort study. Asian Pac J Allergy Im-               serological follow-up with conventional and conformational
      munol. 2019; 39(4):272-278. DOI: 10.12932/AP-231118-0446.                     anti-desmoglein antibodies in treatment-naïve and previously
      PMID: 31175713.                                                               treated patients with pemphigus vulgaris after receiving rit-
46.   Delavarian Z, Layegh P, Pakfetrat A, Zarghi N, Khorashadizadeh                uximab. Int J Womens Dermatol. 2019;5(5):372-377. DOI:
      M, Ghazi A. Evaluation of desmoglein 1 and 3 autoantibodies in                10.1016/j.ijwd.2019.05.008. PMID: 31909160. PMCID:
      pemphigus vulgaris: correlation with disease severity. J Clin Exp             PMC6938821.




8                                                                                           Review | Dermatol Pract Concept. 2022;12(2):e2022116
