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Autoimmune Blistering Disease
- Diagnostic Methodology for Pemphigus and Pemphigoid -
Pemphigus
BP
EBA
Epidermal side
Epidermal side
Dermal side
Dermal side
Epidermal cell-cell junction
BP230
BP230
Anti-BP230
BP180
BP180
Anti-BP180
Dsg3
Anchoring fibril
Anchoring fibril
Dsg1
Blister
NC16a
Complement
www.mblintl.com
Blister
Anti-type VII collagen
Structure of the epidermis
CONTENTS
The epidermis generally consists of four layers: basal
Structure of the epidermis
3
Classification of autoimmune blistering diseases3
layer (stratum basale), spinous layer (stratum spinosum),
(stratum corneum). Keratinocytes are the major component
4
4
layer, the outermost layer of the epidermis. Several
Pemphigus (PV/PF) and anti-desmoglein 1 & 3 IgG autoantibodies
5
types of intercellular junctions in the epidermis, such as
desmosomes and tight junctions, are involved in protection
Pemphigoid
BOX Salt-split skin immunofluorescence
Bullous pemphigoid (BP) and anti-BP180 and anti-BP230 IgG autoantibodies
Epidermolysis bullosa acquisita (EBA) and anti-type VII collagen IgG autoantibodies
Sites of the skin blister formation in BP and EBA
6
against mechanical stress, physical stimulation or infectious
agents. Desmosomes are composed of transmembrane
6
proteins [e.g., desmoglein (Dsg) 1, Dsg3, and desmocollin]
7
and intracellular proteins (e.g., desmoplakin). Desmogleins
Basal layer, the deepest layer of the epidermis, rests
upon the basement membrane zone (BMZ) of dermal-
8
9
Dsg1: desmoglein 1
Epidermal cells
Cornified layer
Plakoglobin
Desmoplakin
Granular layer
Plakophilin
Epidermis
Dermal-epidermal junction
BP230
BP180
Basal cells
Integrin α6β4
Hemi-desmosome
Basal layer
10
Lamina densa
Dermis
Laminin 332
of desmosome, being involved in adhesion in DEJ. Two
integrin α6β4 are transmembrane proteins which link to
BMZ. BP230 (BPAG1) and plectin (HD-1) are cytoplasmic
11
Differential diagnoses of autoimmune blistering diseases with ELISA
proteins involved in the organization of the cytoskeleton.
BMZ is divided into the lamina densa and the lamina lucida.
Screening of autoimmune blistering diseases
11
Differential diagnosis of pemphigus
11
collagen. The lamina lucida contains heparan sulfate
Differential diagnosis of BP
12
proteoglycan, fibronectin and Laminin 332 (laminin 5).
Differential diagnosis of epidermolysis bullosa acquisita (EBA)
12
Dermis collagen
Monitoring disease activities by ELISA
Monitoring disease activity in mucocutaneous PV with
MESACUP Desmoglein ELISA kits13
Monitoring disease activity in BP with MESACUP BP180 TEST
13
Monitoring disease activity in EBA with MESACUP Anti-Type VII collagen TEST
13
Acknowledgements / References
Pemphigus
group
Pemphigus foliaceus, PF
Pemphigus vulgaris, PV
Mucosal dominant type
Pemphigus vegetans (PV subtype)
Herpetiform pemphigus
Paraneoplastic pemphigus, PNP
collagen. Type VII collagen secures the lamina densa to the
IgA pemphigus
(Intraepidermal neutrophilic IgA dermatosis)
Blistering disease is the general term for several diseases
epidermal or epidermal-dermal cohesions.
14
1-3)
Drug-induced pemphigus
Neonatal pemphigus
dermis through association with dermis collagens.
caused by congenital or acquired interruptions of
Brazilian pemphigus (PF endemic type)
Others
the lamina lucida and the lamina densa and connects
with blisters and erosion on the skin and mucous membrane
Pemphigus erythematosus (PF localized type)
Mucocutaneous type
Laminin 332 serves as a major anchoring protein between
13
Classification of autoimmune blistering diseases
The major component of the lamina densa is type IV
BP180 and integrin α6β4 in the lamina lucida with type VII
Anchoring fibril
(Type VII collagen)
Dermis
have hemidesmosome, a structure comparable to a half
pemphigoid antigen 2), or type XVII collagen ] and
Basement membrane zone
Lamina lucida
Basement
membrane zone
hemidesmosomal components, BP180 [BPAG2 (bullous
ELISA kits for the diagnosis of autoimmune blistering diseases
Plectin
Spinous layer
maintain epidermal cohesion in a Ca2+-dependent manner.
8
Desmoglein 1 is a target in bullous infectious diseases
Desmosome
and desmocollins, which are the cadherin family proteins,
8
epidermal junction (DEJ). Keratinocytes in basal layers
TOPICS
Dsg3: desmoglein 3
Epidermal cells
from basal cells to the finally differentiated, cornified
Clinical characterization
Desmocollin
granular layer (stratum granulosum), and cornified layer
Clinical characterization
Epidermal cell-cell junction
of the epidermis. These cells progressively differentiate
Pemphigus
Normal skin
Pemphigoid
group
This
brochure summarizes the clinical manifestations, the
Bullous pemphigoid, BP
mechanism of the blister formation, and the serological
Herpes gestationis, HG (BP subtype)
diagnosis on various autoimmune blistering diseases.
Epidermolysis bullosa acquisita, EBA
Mucous membrane pemphigoid, MMP
Dermatitis herpetiformis [Dühring]
Linear IgA bullous dermatosis, LAD
Anti-p200 pemphigoid
(Anti-laminin γ1 pemphigoid)
Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid -
3
Paraneoplastic pemphigus (PNP)
Pemphigus
Figure 2
Skin lesion
PNP is an autoimmune mucocutaneous disease associated
Histopathology
with underlying malignancy, particularly lymphoproliferative
neoplasms. Painful erosions and ulcerations occur in the
oral mucous membrane. In addition, many patients with
PNP have ocular mucosal lesions and pseudomembranous
Overview
Pemphigus is a group of autoimmune blistering diseases of the skin and mucous membranes which are characterized
histologically by intraepidermal blisters due to acantholysis (i.e., disruption of the intercellular connections between
keratinocytes of the epidermis) and immunopathologically by in vivo bound and circulating immunoglobulin G (IgG)
antibodies directed against the cell surface of keratinocytes. Most common ages of disease onset is about 40 to 60
years. Nikolsky’s sign (i.e., blistering induced by lateral pressure to the normal-appearing skin) is also a characteristic
feature of pemphigus. The target antigens in pemphigus are Dsg1 and 3, 4, 5) members of the cadherin super family. Pemphigus
can be classified into pemphigus vulgaris (PV), pemphigus foliaceus (PF), paraneoplastic pemphigus (PNP), and others.
The blisters in PV and PF occur in the deeper region of the epidermis (just above the basal layer) and the upper layer,
respectively.
conjunctivitis, resulting in ankyloblepharon in severe
cases. 6)
Acantholytic blisters in the epidermis are formed
in the superficial epidermis.
Other types of pemphigus
Herpetiform pemphigus is characterized clinically by
Direct immunofluorescence staining of
the skin taken from a patient with PF
erythematous urticarial plaques and vesicles that present
in a herpetiform arrangement, histologically eosinophilic
spongiosis with minimal or no acantholysis, and serologically
IgG autoantibodies against cell surfaces of keratinocytes.
Clinical characterization
Drug-induced pemphigus is induced by drugs such as
Figure 1
Oral lesion
Histopathology
Pemphigus vulgaris (PV)
D-penicillamine or captopril. Neonatal pemphigus is a
disease that rarely occurs in infants born to mothers with
PV. IgA pemphigus is characterized by tissue-bound and
PV is the most common of pemphigus diseases. The
circulating IgA autoantibodies that target the desmosomal
majority of patients have painful mucous membrane
proteins or unidentified cell surface antigens in the epidermis.
IgG deposits on the cell surface of the epidermis,
and stronger staining in the upper layers of the
epidermis than the lower layers.
erosions, especially in the oral cavity (Figure 1). While
mucous membranes are mainly affected in mucosal
dominant PV (MDPV), in mucocutaneous PV (MCPV),
Pemphigus (PV/PF) and anti-desmoglein 1 & 3
IgG autoantibodies
blisters and erosions are not only present on the mucosal
area but also on the skin, predominantly the regions
Acantholytic blisters in the epidermis are formed
just above the basal layer.
prone to pressure and friction, such as scalp, axilla,
groin, upper part of back, and buttock. Pemphigus
Skin lesions
vegetans, a rare form of PV, is characterized by vegetating
granulomatous lesions.
Figure 3
Dsg1
Dsg1 and Dsg3, the pemphigus target antigens, have
different intraepidermal expression patterns in the skin
and mucous membranes (Figure 3). In the skin, Dsg1 is
Dsg3
Anti-Dsg1
Anti-Dsg3
Pemphigus vulgaris（PV, Mucosal dominant type)
Mucosal lesion: Suprabasal epithelia
Skin lesion: None or only localized
Dsg1
Dsg1
distributed throughout the epidermis, but more strongly
in the superficial layers, whereas Dsg3 is expressed
in the lower part of the epidermis (basal or parabasal
Pemphigus foliaceus (PF)
layers). In the mucous membranes, on the other hand,
PF is characterized by scaly crusted erosions. These
Dsg1 and Dsg3 are expressed throughout the mucous
lesions are scattered in seborrheic regions such as the
membrane, but the expression level of Dsg1 is much
scalp, face, and upper trunk, while the mucous membranes
lower than that of Dsg3. The clinical features of pemphigus
are never affected (Figure 2). Symptoms of patients with
can be explained by “desmoglein compensation theory”,
PF are generally not as serious compared to those of PV.
i.e., these antigens can compensate their adhesive
Pemphigus erythematosus (Senear-Usher syndrome), the
Direct immunofluorescence staining of the skins taken from patients with PV
Dsg3
Dsg3
Dsg3 function suppressed
Dsg3 function suppressed
Pemphigus vulgaris (PV, Mucocutaneous type)
Skin lesion: Suprabasal epidermis
Dsg1
Mucosal lesion: Suprabasal epithelia
Dsg1
functions when they are co-expressed in the same cells.
localized form of PF, is associated with butterfly rash on
16, 17)
the face. Fogo selvagem (Brazilian pemphigus foliaceus)
present, the blister formations occur only in the deep
is the endemic type of PF in the area of South America,
layers of mucous membranes that lack the compensation
especially Brazil.
by Dsg1 (mucosal-dominant type of PV). Patients who
In cases of PV, when only anti-Dsg3 antibodies are
Dsg3
Both Dsg1 & Dsg3 function suppressed
Pemphigus foliaceus (PF)
Skin lesion: Superficial epidermis
have both anti-Dsg1 and anti-Dsg3 antibodies, the
blisters are formed in mucous membranes as well as
Dsg3
Both Dsg1 & Dsg3 function suppressed
Dsg1
Mucosal lesion: None
Dsg1
the skin (mucocutaneous type of PV). On the other hand,
Mucosal dominant PV
IgG deposits on the cell surface of the
epidermis, and stronger staining in the deeper
layers than the upper layers.
Mucocutaneous PV
IgG deposits on the cell surface throughout
the epidermis.
in cases where antibodies are present only against Dsg1,
the blisters are formed only in the upper epidermis of
skin, where Dsg1 is present without compensation by
Dsg3
Dsg1 function suppressed
Dsg3
Dsg1 function suppressed
Dsg3 (PF). 9)
4 Autoimmune Blistering Diseases
- Diagnostic Methodology for Pemphigus and Pemphigoid -
Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid -
5
Epidermolysis bullosa acquisita (EBA)
Pemphigoid
EBA is a subepidermal autoimmune skin disease
Figure 5
Skin lesion
Histopathology
associated with autoimmunity to type VII collagen (Figure
5), which is the major component of anchoring fibrils.
The classic presentation of EBA is noninflammatory
blistering on the extremities that heal with scarring and
Overview
Pemphigoid is a group of diseases characterized histologically by subepidermal blisters and immunopathologically by
linear deposition of IgG and complement C3 at basement membrane zone (BMZ) in the skin from patients with
circulating IgG against the molecules within the dermal-epidermal junction (DEJ). The target antigens recognized by
autoantibodies in patients with bullous pemphigoid (BP) are BP180 (a 180 kDa transmembrane protein), and BP230
(a 230 kDa intracellular protein). The target antigens recognized by autoantibodies in other diseases of this group
include type VII collagen in epidermolysis bullosa acquisita (EBA); laminin 332 (laminin 5, epilligrin), one of the target
antigens in mucous membrane pemphigoid (MMP); a 97 kDa protein (BP180 degradation product) in linear IgA bullous
dermatoses (LAD); and laminin γ1 in anti-p200 pemphigoid. 10−12).
Clinical characterization
membrane. Histopathology shows subepidermal blisters
Sub-epidermal blister formation.
with different degrees of inflammation and neutrophil
infiltration. Direct immunofluorescence assay reveals
Indirect immunofluorescence assay
IgG deposition along BMZ. 15, 16)
Dermatitis herpetiformis (Dühring disease)
Dermatitis herpetiformis (DH) is characterized by chronic
eruptions typically on elbows, knees, and back with intense
with significant pruritus. Histopathological analysis
pruritus. The granular deposits of IgA are detected in the
detects subepidermal blisters and superficial dermal
IgG deposits in BMZ.
papillary dermis by direct immunofluorescence assay.
infiltrations of eosinophils, lymphocytes, and macrophages
Circulating IgA autoantibodies against transglutaminase
(Figure 4). Mucous membrane erosions are occasionally
have been identified in patients. However, the exact
found in some patients with BP. BP usually occurs in
mechanisms on the IgA deposition in the skin remain
elderly individuals.
Salt-split skin immunofluorescence
unclear. In addition, it has been reported that the IgA
Herpes gestationis (HG, pemphigoid gestationis), a
13)
observed on any part of the body including the mucous
Skin lesion
The skin lesions of BP are characterized by tense blisters
postpartum period.
varied and inflammatory blisters and eruptions can also be
Figure 4
Bullous pemphigoid (BP)
subtype of BP, occurs during pregnancy and the immediate
milia formation.However, the clinical manifestation is
Histopathology
Direct immunofluorescence
Histopathology of HG shows
deposits may be reduced in the patients who go on a
long-term gluten-free diet.
17)
subepidermal blisters with eosinophil infiltration.
The localization of autoantibodies of EBA is distinct from the deposition in BP. To
differentiate EBA from BP, salt-split skin technique (i.e., skin incubated with 1 M NaCl to
fracture dermal-epidermal junctions) followed by direct immunofluorescence assay is
allowed to discriminate EBA from BP. The antibodies from the EBA patients are localized
at the dermal side of the separation.
BP
Mucous membrane pemphigoid (MMP)
Linear IgA dermatosis (LAD)
MMP had been called cicatricial pemphigoid due to
LAD is a rare blistering disease with the onset over 40
scar formation after blisters and erosions. However,
not all of the patients have scarring. MMP is a rare
years or under 10 years of age [chronic bullous diseases
Subepidermal blister formation.
Linear deposition of IgG along BMZ.
of childhood (CBDC)]. The clinical manifestations of
autoimmune blistering disease, involving oral and ocular
LAD are itchy erythematous papules and blisters. Direct
mucous membranes. Linear depositions of IgG and/or
immunofluorescence of perilesional skin from the patient
complement C3 along BMZ can be detected by direct
with LAD reveals IgA linear deposition along BMZ. A
immunofluorescence assay. A major MMP target autoantigen
target antigen is the 97 kDa protein, an extracellular
is BP180. Other target antigens include laminin 332 and
domain of BP180. 18)
BP230.
IgG binds to the epidermal side of
the split skin
(indicated by arrow).
EBA
14)
Anti-p200 pemphigoid (Anti-laminin γ1 pemphigoid)
IgG binds to the dermal side of
the split skin
(indicated by arrow).
Anti-p200 pemphigoid is a blistering skin disease
occasionally seen in patients with psoriasis. Recently,
laminin γ1 was identified as the target antigen. 12)
6 Autoimmune Blistering Diseases
- Diagnostic Methodology for Pemphigus and Pemphigoid -
Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid -
7
Pemphigoid
Topic
Sites of the skin blister formation in BP and EBA
Bullous pemphigoid (BP) and anti-BP180 and
anti-BP230 IgG autoantibodies
BP
Anti-BP180 autoantibodies in patients with BP are
Staphylococcal scalded skin syndrome (SSSS) and bullous impetigo are blistering diseases caused by exotoxins (exfoliative
generally considered to be pathogenic. In vitro studies
toxin, ET) produced by Staphylococcus aureus. SSSS is common in newborns, infants, and patients with renal failure or
indicate that BP IgG activates the classical complement
immunodeficiency. It is considered that ET disseminated through the bloodstream causes erythema and blisters throughout
pathway, thereby causing activation of inflammatory cells
with the degranulation and resulting dermal-epidermal
separation.
19)
the body. Nikolsky’s sign is also detected. In bullous impetigo, however, ET produces the blisters locally at the infected area.
Epidermal side
The mechanisms of blister formation in both diseases had remained unclear until recently, although classic studies showed that
However, some reports suggested the
Anti-BP230
non-inflammatory mechanism of the blister formation;
20)
ET could induce blisters in neonatal mice in the 1970’s.
Dermal side
anti-BP180 antibodies cause conformational changes
of BP180, to reduce functional BP180 molecules.
- Desmoglein 1 is a target in bullous infectious diseases -
SSSS and pemphigus foliaceus (PF) share many similar features; (1) only the skin is affected, but not the mucous membrane,
The
(2) the blister formation occurs in the upper epidermis, (3) no necrotic keratinocytes precede the blisters, and (4) injections
autoantibodies from most patients with BP recognize the
Anti-BP180
NC16a domain, which is a non-collageneous region of
indicated that several ET subtypes (exfoliative toxin A, B, and D) induced the blister formation by a cleavage of Dsg1 at Gul381
BP180, located just outside the cell membrane.
In contrast, anti-BP230 antibodies may not directly
through their serine protease activity, while they do not cleave Dsg3. This reflects the histological manifestation in SSSS, of
BP230
which the lesion is localized in the skin. It is clinically significant that the inactivation of Dsg1 causes superficial skin blisters in
cause the blister formation because it is unlikely that the
antibodies can access to BP230 localized in the cytoplasm.
Nevertheless, anti-BP230 antibodies are a useful diagnostic
marker for BP with high specificity. Anti-BP230 antibodies
of ET into neonatal mice induce superficial blisters whose histology is identical to PF. In addition, various experiments
two different skin diseases, PF and SSSS. 23,
Blister
24)
BP180
are detected only in some patients with BP. 21)
Anchoring fibril
NC16a
Complement
Epidermolysis bullosa acquisita (EBA) and
anti-type VII collagen IgG autoantibodies
EBA
Autoantibodies against type VII collagen are associated
with dysfunction of anchoring fibrils in dermal-epidermal
junctions. Direct immunofluorescence staining shows
Epidermal side
IgG deposition within the sub-lamina densa of the skin.
The localization of autoantibodies is distinct from the
deposition in BP. To differentiate EBA from BP, salt-split
Dermal side
skin technique (i.e., see page 7) followed by direct
immunofluorescence assay is allowed to discriminate
EBA from BP. The antibodies from the EBA patients are
localized at the dermal side of the separation.
Type VII collagen is composed of three identical alpha-
BP230
chains (290 kDa). Each chain consists of a 145 kDa
ET
non-collagen (NC1) domain, a typical collageneous
domain, and a 34 kDa non-collagen (NC2) domain.
Epitopes recognized by autoantibodies from EBA
BP180
Blister
Dsg1
patients are mainly on NC1. NC2 is also considered to
contain minor epitopes. 22)
ET: Exfoliative toxin
Anchoring fibril
Anti-type VII collagen
8 Autoimmune Blistering Diseases
- Diagnostic Methodology for Pemphigus and Pemphigoid -
Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid -
9
ELISA kits for the diagnosis of autoimmune blistering diseases
Differential diagnoses of autoimmune blistering diseases with ELISA
Interpretation of results with MESACUP Anti-Skin Profile TEST*
Screening of autoimmune blistering diseases
DSG 1
Profile Kit
BP180
DSG 3
Type VII collagen
BP230
ELISA Index (U/mL)
MESACUP Anti-Skin Profile ELISA TEST* is an ELISA
kit for the qualitative detection of IgG antibodies to Dsg1,
Dsg3, BP180, BP230 and type VII collagen in human
MESACUP Anti-Skin Profile TEST*
1
2
3
4
5
6
7
8
Pemphigus
EBA
E: BP180
F: BP230
G: Type VII collagen
H: Assay control
(Human IgG)
epidermis side
epidermis side
dermis side
dermis side
Code No.
Disconcordance
Concordance Rate (%)
189
3
98.4
Dsg3
191
1
99.5
BP180
191
1
99.5
BP230
189
3
98.4
Type VII collagen
189
3
98.4
Interpretation of results with MESACUP Desmoglein ELISA kits
Differential diagnosis of pemphigus
ELISA Index (U/mL)
Positive
MESACUP-2 TEST Desmoglein 1†
≥ 20
MESACUP-2 TEST Desmoglein 3†
≥ 20
MESACUP Dsg1 & Dsg3 ELISA TEST SYSTEM††
≥ 20
staining patterns between PV and PF according to the
tissue distribution of Dsg1 and 3 (see page 4). However,
Product
Volume
Storage
Antigens / Description
the immunofluorescence patterns of tissues are difficult
to clearly discriminate between the two diseases.
RG-M7593-D
MESACUP Dsg1 & Dsg3 ELISA TEST SYSTEM
48 wells
2-8 °C
Recombinant human Dsg1 and Dsg3
RG-M7612-D
MESACUP BP180 TEST
48 wells
2-8 °C
Recombinant human BP180 NC16a
RG-M7613-D
MESACUP BP230 TEST
48 wells
2-8 °C
Recombinant human BP230 (NT and CT)
RG-7845R2
Anti-Type VII collagen ELISA Kit (For Research use only)
48 wells
2-8 °C
Recombinant human type VII collagen NC1 and NC2
RG-7115R
Anti-Skin Profile ELISA Kit (For Research use only)
8 wells x 12
2-8 °C
Recombinant human Dsg1, Dsg3, BP180, BP230 and type VII collagen
†
For EU Customers only
For U.S. Customers only
††
MESACUP Desmoglein ELISA kits are for measurements
of anti-Dsg1 and anti-Dsg3 antibodies in serum, using the
Disease classification of pemphigus by anti-Dsg antibody profiling
recombinant Dsg1 or Dsg3 as the solid-phase antigen.
These ELISA kits provide sensitive and specific assays
Types of pemphigus
Anti-Dsg3 antibody
Anti-Dsg1 antibody
for the differential serological diagnosis between PV and
Mucosal-dominant PV
Positive
Negative
PF on the basis of their characteristic profiles of auto
Mucocutaneous PV
Positive
Positive
PF
Negative
Positive
antibodies. 25)
Presence of anti-Dsg1 and anti-Dsg3 antibodies in PV, PF, BP, and
normal sera
For EU customers
Code No.
Concordance
Dsg1
Immunofluorescence assay may reveal differences in
For US customers
≥ 15
Comparison of existing individual ELISA kits for autoimmune
blistering diseases and MESACUP Anti-Skin Profile TEST*
simultaneously in a single assay.
9 10 11 12
A
B
C
D
E
F
G
H
A: Calibrator 1 (0 U/mL)
B: Ca librator 2 (100 U/mL)
C: Dsg1
D: Dsg3
Bullous pemphigoid
Positive
< 15
Value was determined by analysis with 30 PV, 30 PF, 60 BP, 30 EBA and 42 normal samples.
serum. These anti-skin autoantibodies can be detected
Detects 5 anti-skin antibodies
in a single assay
Negative
Product
Volume
Storage
Antigens / Description
Anti-Dsg1
Anti-Dsg3
250
MESACUP-2 TEST Desmoglein 1
48 wells
2-8 °C
Recombinant human Dsg1
RG-7885EC-D
MESACUP-2 TEST Desmoglein 3
48 wells
2-8 °C
Recombinant human Dsg3
RG-7695EC-D
MESACUP BP180 TEST
48 wells
2-8 °C
Recombinant human BP180 NC16a
RG-7613EC-D
MESACUP BP230 TEST
48 wells
2-8 °C
Recombinant human BP230 (NT and CT)
RG-7845E
MESACUP Anti-Type VII collagen TEST
48 wells
2-8 °C
Recombinant human type VII collagen NC1 and NC2
200
ELISA Index
RG-7880EC-D
150
100
50
20
20
RG-7115EC-D
MESACUP Anti-Skin Profile TEST
10 Autoimmune Blistering Diseases
- Diagnostic Methodology for Pemphigus and Pemphigoid -
8 wells x 12
2-8 °C
0
Recombinant human Dsg1, Dsg3, BP180, BP230 and type VII collagen
PV
PF
(27/39) (31/31)
*U.S. Customers : for research use only (Not for use in diagnostic procedures)
BP
(0/45)
Normal
(0/180)
PV
PF
(39/39) (0/31)
BP
(1/45)
Normal
(0/180)
Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid -
11
Differential diagnoses of autoimmune blistering diseases with ELISA
Interpretation of results with MESACUP BP180 and MESACUP BP230
Differential diagnosis of BP
ELISA Index (U/mL)
MESACUP BP180 TEST is an ELISA kit for the detection
of anti-BP180 antibodies in human serum, using the
antigen. The sensitivity of the kit is 84% (54/64) in patients
Negative
Positive
MESACUP BP180 TEST *1
<9
≥9
MESACUP BP230 TEST *
<9
≥9
2
*1: Values were established by ROC analysis with 64 BP samples and control samples (42 PF, 69 PV and 336 normal samples).
*2: Values were established by ROC analysis with 72 BP samples and 109 normal samples.
Clinical sensitivity of anti-BP180 and anti-BP230 ELISA for BP sera†
MESACUP BP230 TEST is an ELISA kit using both
the recombinant N-terminal and C-terminal domains
Active stage
Remission
Total
of BP230 for detection of anti-BP230 antibodies in
MESACUP BP180 TEST
84% （54 / 64）
65% （113 / 175）
70% （167 / 239）
human serum. The sensitivity is 58% (37/64) in patients
MESACUP BP230 TEST
58% （37 / 64）
78% （136 / 175）
72% （173 / 239）
diagnosed with BP in the active stage. Most significantly,
MESACUP BP180 TEST
+
MESACUP BP230 TEST
†
combining the results of both BP180 and BP230 ELISA
that amounts of anti-Dsg1 and anti-Dsg3 antibodies
decrease during the improvement, correlating with the
improvement of cutaneous erosions. Amounts of
autoantibodies also correspond to the pemphigus disease
area index (PDAI).
94% （60 / 64）
98% （172 / 175）
97% （232 / 239）
In recent studies, the amount of anti-Dsg1 and anti-Dsg3
intravenous immunoglobulin (IVIG) therapies.
27)
29-31)
Presence of anti-BP180 and anti-BP230 antibodies in BP, PV/PF,
and normal sera
autoantibodies in human serum. Interestingly, 96%
(47/49) of EBA sera were positive with this kit, and none of
20 BP sera were positive. Thus, our ELISA kit provides a
sensitive and specific assay for the differential serological
diagnosis of EBA and BP.
28)‡
150
Anti-Dsg 1
* PDAI: Pemphigus Disease Area Index
0
14
37
Several clinical studies suggested that the data using
MESACAP BP180 TEST demonstrated that the
100
disappearance of blisters correlated with decrease in the
amount of anti-BP180 antibody. 27,33,34)
50
1000
Additionally, in a previous report on one patient with
0
50
40
30
20
10
0
Clinical manifestation of BP disease and anti-BP180 antibody levels
9
BP
BP
PV/PF
(Active stage) (Remission)
(1/94)
(54/64) (113/175)
Normal
(5/336)
9
BP
BP
PF/PV
(Active stage) (Remission)
(1/94)
(37/64) (135/175)
Normal
(0/109)
herpes gestationis (HG), the amount of anti-BP180
antibodies was consistent with the severity of the
erythema and blisters of mothers and neonates in the
Interpretation of results with MESACUP Anti-Type VII collagen TEST*
ELISA Index (U/mL)
MESACUP Anti-Type VII collagen TEST
Negative
Positive
<6
≥6
peripartum period.
35)
Anti-BP180 antibodies
(U/mL)
collagen for measurement of anti-type VII collagen
Monitoring disease activity in BP
with MESACUP BP180 TEST
200
ELISA Index
using the recombinant NC1 and NC2 domains of type VII
Days after treatment
200
Anti-BP230
Anti-BP180
MESACUP Anti-Type VII collagen TEST is an ELISA kit
Anti-Dsg 3
400
Days after treatment
250
Differential diagnosis of epidermolysis bullosa
acquisita (EBA)
600
0
antibodies decreased significantly after rituximab and
Data courtesy of Kobayashi M, Amagai M, Kuroda-Kinoshita K, Hashimoto T, Shirakata Y, Hashimoto K, Nishikawa T. BP180
ELISA using bacterial recombinant NC16a protein as a diagnostic and monitoring tool for bullous pemphigoid. J. Dermatol. Sci.
30: 224-32, 2006, PubMed: 12443845
PDAI
kits.
Clinical manifestation of PV disease and anti-desmoglein antibody
levels
Clinical data using MESACUP Desmoglein kits suggest
with BP in the active stage. 26)
higher clinical sensitivity (97%) can be achieved by
Monitoring disease activity in mucocutaneous
PV with MESACUP Desmoglein ELISA kits
Anti-desmoglein
antibodies
(U/mL)
recombinant BP180 NC16a protein as the immobilized
Monitoring disease activities by ELISA
800
600
400
200
0
These findings strongly suggest
0
12
28
35
Days after treatment
the clinical importance of periodic measurements of the
autoantibodies in patients with BP.
Values were determined by ROC analysis with 49 EBA, 20 PF, 20 BP and 266 normal samples.
Presence of anti-type VII collagen antibodies in EBA, PV, BP, and
normal sera ‡
with the improvement of cutaneous symptoms in a patient
with EBA after treatment. 28, 36, 37)
300
Anti-type VII collagen antibodies
(U/mL)
200
100
6
0
EBA
PV
(47/49) (0/20)
12 Autoimmune Blistering Diseases
- Diagnostic Methodology for Pemphigus and Pemphigoid -
BP
(0/20)
Normal
(1/266)
*U.S. Customers : for research use only (Not for use in diagnostic procedures)
Data for EU Customers only.
‡
Clinical manifestation of EBA disease and anti-type VII collagen
antibody levels
The amount of anti-type VII collagen antibodies decreased
400
ELISA Index
Monitoring disease activity in EBA
with MESACUP Anti-Type VII collagen TEST*
6000
4000
2000
0
0
27
56
75
Days after treatment
*U.S. Customers : for research use only (Not for use in diagnostic procedures)
Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid -
13
Acknowledgements
We are grateful to Drs. Masayuki Amagai and Jun Yamagami of Keio University School of Medicine for data and valuable
advice, and to Dr. Ken Ishii of Teikyo University Chiba Medical Center and Dr. Takashi Hashimoto of Kurume University School of
Medicine for data.
21. Kasperkiewicz M, Zillikens D. The pathophysiology of bullous pemphigoid. Clin. Rev. Allergy Immunol. 33: 67–77, 2007, PubMed: 18094948
22. Ishii N, Yoshida M, Hisamatsu Y, Ishida-Yamamoto A, Nakane H, Iizuka H, Tanaka T, Hashimoto T. Epidermolysis bullosa acquisita sera react
with distinct epitopes on the NC1 and NC2 domains of type VII collagen: study using immunoblotting of domain-specific recombinant proteins and
postembedding immunoelectron microscopy. Br. J. Dermatol. 150: 843−851, 2004, PubMed: 15149495
23. Amagai M, Matsuyoshi N, Wang ZH, Andl C, Stanley JR. Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1.
Nat. Med. 6: 1275−1277, 2000, PubMed: 11062541
24. Amagai M, Yamaguchi T, Hanakawa Y, Nishifuji K, Sugai M, Stanley JR. Staphylococcal exfoliative toxin B specifically cleaves desmoglein 1. J. Invest.
Dermatol. 118: 845−850, 2002, PubMed: 11982763
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14 Autoimmune Blistering Diseases
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Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid -
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