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Autoimmune Blistering Disease- Diagnostic Methodology for Pemphigus and Pemphigoid -

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EBABP

Epidermal side

Dermal side

Epidermal side

Dermal side

Dsg3

Dsg1

Epidermal cell-cell junction

ComplementNC16a

Blister

Anti-BP230

Anti-BP180

Blister

Anti-type VII collagen

Anchoring fibril

BP230

BP180

BP230BP230

BP180BP180

Anchoring fibril

BP230

BP180

Pemphigus

Structure of the epidermis

The epidermis generally consists of four layers: basal

layer (stratum basale), spinous layer (stratum spinosum),

granular layer (stratum granulosum), and cornified layer

(stratum corneum). Keratinocytes are the major component

of the epidermis. These cells progressively differentiate

from basal cells to the finally differentiated, cornified

layer, the outermost layer of the epidermis. Several

types of intercellular junctions in the epidermis, such as

desmosomes and tight junctions, are involved in protection

against mechanical stress, physical stimulation or infectious

agents. Desmosomes are composed of transmembrane

proteins [e.g., desmoglein (Dsg) 1, Dsg3, and desmocollin]

and intracellular proteins (e.g., desmoplakin). Desmogleins

and desmocollins, which are the cadherin family proteins,

maintain epidermal cohesion in a Ca2+-dependent manner.

Basal layer, the deepest layer of the epidermis, rests

upon the basement membrane zone (BMZ) of dermal-

epidermal junction (DEJ). Keratinocytes in basal layers

have hemidesmosome, a structure comparable to a half

of desmosome, being involved in adhesion in DEJ. Two

hemidesmosomal components, BP180 [BPAG2 (bullous

pemphigoid antigen 2), or type XVII collagen] and

integrin α6β4 are transmembrane proteins which link to

BMZ. BP230 (BPAG1) and plectin (HD-1) are cytoplasmic

proteins involved in the organization of the cytoskeleton.

BMZ is divided into the lamina densa and the lamina lucida.

The major component of the lamina densa is type IV

collagen. The lamina lucida contains heparan sulfate

proteoglycan, fibronectin and Laminin 332 (laminin 5).

Laminin 332 serves as a major anchoring protein between

the lamina lucida and the lamina densa and connects

BP180 and integrin α6β4 in the lamina lucida with type VII

collagen. Type VII collagen secures the lamina densa to the

dermis through association with dermis collagens.

Blistering disease is the general term for several diseases

with blisters and erosion on the skin and mucous membrane

caused by congenital or acquired interruptions of

epidermal or epidermal-dermal cohesions. 1-3) This

brochure summarizes the clinical manifestations, the

mechanism of the blister formation, and the serological

diagnosis on various autoimmune blistering diseases.

Cornified layer

Spinous layer

Granular layer

Basal layer

Epidermis

Basement membrane zone

Integrin α6β4

Dermis collagen

BP230BP230Plectin

Anchoring fibril(Type VII collagen)Anchoring fibril(Type VII collagen)

Desmocollin

Dsg3: desmoglein 3

Dsg1: desmoglein 1

Desmocollin

Dsg3: desmoglein 3

Dsg1: desmoglein 1

Laminin 332

Plakoglobin

Plakophilin

Desmoplakin

Epidermal cells

Epidermal cells

Epidermal cells

Epidermal cells

Basal cellsBasal cells

Lamina lucida

Lamina densa

Lamina lucida

Lamina densa

Dermis

DermisDermis

Desmosome

Hemi-desmosome

Desmosome

Hemi-desmosome

Basement membrane zoneBasement membrane zone

BP180BP180

Pemphigusgroup

Pemphigoidgroup

Pemphigus vulgaris, PV

Paraneoplastic pemphigus, PNP

Herpetiform pemphigus

Drug-induced pemphigus

Neonatal pemphigus

IgA pemphigus (Intraepidermal neutrophilic IgA dermatosis)

Bullous pemphigoid, BP Epidermolysis bullosa acquisita, EBA

Mucous membrane pemphigoid, MMP Dermatitis herpetiformis [Dühring]

Linear IgA bullous dermatosis, LAD Anti-p200 pemphigoid(Anti-laminin γ1 pemphigoid)

Pemphigus foliaceus, PF

Pemphigus erythematosus (PF localized type)

Brazilian pemphigus (PF endemic type)

Herpes gestationis, HG (BP subtype)

Pemphigus vegetans (PV subtype)

Mucosal dominant type

Mucocutaneous type

Others

Classification of autoimmune blistering diseases

Normal skin Epidermal cell-cell junction

Dermal-epidermal junction

3Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid -

Structure of the epidermis

Classification of autoimmune blistering diseases 3

Pemphigus

Clinical characterization 4

Pemphigus (PV/PF) and anti-desmoglein 1 & 3 IgG autoantibodies 5

Pemphigoid

Clinical characterization 6

Salt-split skin immunofluorescence 7

Bullous pemphigoid (BP) and anti-BP180 and anti-BP230 IgG autoantibodies 8

Epidermolysis bullosa acquisita (EBA) and anti-type VII collagen IgG autoantibodies 8

Sites of the skin blister formation in BP and EBA 8

ELISA kits for the diagnosis of autoimmune blistering diseases

Differential diagnoses of autoimmune blistering diseases with ELISA

Screening of autoimmune blistering diseases 11

Differential diagnosis of pemphigus 11

Differential diagnosis of BP 12

Differential diagnosis of epidermolysis bullosa acquisita (EBA) 12

Monitoring disease activities by ELISA

Monitoring disease activity in mucocutaneous PV with MESACUP Desmoglein ELISA kits 13

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

BOX

3

4

6

11

13

14

C O N T E N T S

TOPICS 9Desmoglein 1 is a target in bullous infectious diseases

10

Pemphigus

4 Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid - 5Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid -

Overview

Pemphigus is a group of autoimmune blistering diseases of the skin and mucous membranes which are characterizedhistologically 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 characteristicfeature of pemphigus. The target antigens in pemphigus are Dsg1 and 3, 4, 5) members of the cadherin super family. Pemphiguscan 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.

Clinical characterization

PV is the most common of pemphigus diseases. The

majority of patients have painful mucous membrane

erosions, especially in the oral cavity (Figure 1). While

mucous membranes are mainly affected in mucosal

dominant PV (MDPV), in mucocutaneous PV (MCPV),

blisters and erosions are not only present on the mucosal

area but also on the skin, predominantly the regions

prone to pressure and friction, such as scalp, axilla,

groin, upper part of back, and buttock. Pemphigus

vegetans, a rare form of PV, is characterized by vegetating

granulomatous lesions.

Pemphigus vulgaris (PV)

PF is characterized by scaly crusted erosions. These

lesions are scattered in seborrheic regions such as the

scalp, face, and upper trunk, while the mucous membranes

are never affected (Figure 2). Symptoms of patients with

PF are generally not as serious compared to those of PV.

Pemphigus erythematosus (Senear-Usher syndrome), the

localized form of PF, is associated with butterfly rash on

the face. Fogo selvagem (Brazilian pemphigus foliaceus)

is the endemic type of PF in the area of South America,

especially Brazil.

Pemphigus foliaceus (PF)

PNP is an autoimmune mucocutaneous disease associated

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

conjunctivitis, resulting in ankyloblepharon in severe

cases. 6)

Paraneoplastic pemphigus (PNP)

Herpetiform pemphigus is characterized clinically by

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.

Drug-induced pemphigus is induced by drugs such as

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

circulating IgA autoantibodies that target the desmosomal

proteins or unidentified cell surface antigens in the epidermis.

Other types of pemphigus

Oral lesion Histopathology

Acantholytic blisters in the epidermis are formed just above the basal layer.

Mucosal dominant PVIgG deposits on the cell surface of the epidermis, and stronger staining in the deeper layers than the upper layers.

Mucocutaneous PVIgG deposits on the cell surface throughout the epidermis.

Skin lesions

Direct immunofluorescence staining of the skins taken from patients with PV

Skin lesion Histopathology

IgG deposits on the cell surface of the epidermis,and stronger staining in the upper layers of the epidermis than the lower layers.

Acantholytic blisters in the epidermis are formed in the superficial epidermis.

Direct immunofluorescence staining ofthe skin taken from a patient with PF

Figure 1

Figure 2

Figure 3

Pemphigus vulgaris （PV, Mucosal dominant type)

Dsg1

Dsg3

Dsg3 function suppressed Dsg3 function suppressed

Dsg1 function suppressed Dsg1 function suppressed

Both Dsg1 & Dsg3 function suppressed Both Dsg1 & Dsg3 function suppressed

Dsg1

Dsg3

Pemphigus foliaceus (PF)

Dsg1

Dsg3

Dsg1

Dsg3

Pemphigus vulgaris (PV, Mucocutaneous type)

Dsg1

Dsg3

Dsg1

Dsg3

Anti-Dsg3Anti-Dsg1Dsg1 Dsg3

Skin lesion: None or only localized Mucosal lesion: Suprabasal epithelia

Skin lesion: Suprabasal epidermis Mucosal lesion: Suprabasal epithelia

Skin lesion: Superficial epidermis Mucosal lesion: None

Pemphigus (PV/PF) and anti-desmoglein 1 & 3 IgG autoantibodies

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

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

layers). In the mucous membranes, on the other hand,

Dsg1 and Dsg3 are expressed throughout the mucous

membrane, but the expression level of Dsg1 is much

lower than that of Dsg3. The clinical features of pemphigus

can be explained by “desmoglein compensation theory”,

i.e., these antigens can compensate their adhesive

functions when they are co-expressed in the same cells. 16, 17) In cases of PV, when only anti-Dsg3 antibodies are

present, the blister formations occur only in the deep

layers of mucous membranes that lack the compensation

by Dsg1 (mucosal-dominant type of PV). Patients who

have both anti-Dsg1 and anti-Dsg3 antibodies, the

blisters are formed in mucous membranes as well as

the skin (mucocutaneous type of PV). On the other hand,

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 (PF). 9)

Pemphigoid


Salt-split skin immunofluorescence

BP

IgG binds to the epidermal side of the split skin(indicated by arrow).

EBA

IgG binds to the dermal side of the split skin(indicated by arrow).

Overview

Pemphigoid is a group of diseases characterized histologically by subepidermal blisters and immunopathologically bylinear deposition of IgG and complement C3 at basement membrane zone (BMZ) in the skin from patients withcirculating IgG against the molecules within the dermal-epidermal junction (DEJ). The target antigens recognized byautoantibodies 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 groupinclude 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 bullousdermatoses (LAD); and laminin γ1 in anti-p200 pemphigoid. 10−12).

MMP had been called cicatricial pemphigoid due to

scar formation after blisters and erosions. However,

not all of the patients have scarring. MMP is a rare

autoimmune blistering disease, involving oral and ocular

mucous membranes. Linear depositions of IgG and/or

complement C3 along BMZ can be detected by direct

immunofluorescence assay. A major MMP target autoantigen

is BP180. Other target antigens include laminin 332 and

BP230. 14)

Mucous membrane pemphigoid (MMP)

EBA is a subepidermal autoimmune skin disease

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

milia formation.However, the clinical manifestation is

varied and inflammatory blisters and eruptions can also be

observed on any part of the body including the mucous

membrane. Histopathology shows subepidermal blisters

with different degrees of inflammation and neutrophil

infiltration. Direct immunofluorescence assay reveals

IgG deposition along BMZ. 15, 16)

Epidermolysis bullosa acquisita (EBA)

Dermatitis herpetiformis (DH) is characterized by chronic

eruptions typically on elbows, knees, and back with intense

pruritus. The granular deposits of IgA are detected in the

papillary dermis by direct immunofluorescence assay.

Circulating IgA autoantibodies against transglutaminase

have been identified in patients. However, the exact

mechanisms on the IgA deposition in the skin remain

unclear. In addition, it has been reported that the IgA

deposits may be reduced in the patients who go on a

long-term gluten-free diet. 17)

Dermatitis herpetiformis (Dühring disease)

LAD is a rare blistering disease with the onset over 40

years or under 10 years of age [chronic bullous diseases

of childhood (CBDC)]. The clinical manifestations of

LAD are itchy erythematous papules and blisters. Direct

immunofluorescence of perilesional skin from the patient

with LAD reveals IgA linear deposition along BMZ. A

target antigen is the 97 kDa protein, an extracellular

domain of BP180. 18)

Linear IgA dermatosis (LAD)

Anti-p200 pemphigoid is a blistering skin disease

occasionally seen in patients with psoriasis. Recently,

laminin γ1 was identified as the target antigen. 12)

Anti-p200 pemphigoid (Anti-laminin γ1 pemphigoid)

Skin lesion Histopathology

IgG deposits in BMZ.

Sub-epidermal blister formation.

Indirect immunofluorescence assay

Figure 4

Figure 5

Clinical characterization

The skin lesions of BP are characterized by tense blisters

with significant pruritus. Histopathological analysis

detects subepidermal blisters and superficial dermal

infiltrations of eosinophils, lymphocytes, and macrophages

(Figure 4). Mucous membrane erosions are occasionally

found in some patients with BP. BP usually occurs in

elderly individuals.

Herpes gestationis (HG, pemphigoid gestationis), a

subtype of BP, occurs during pregnancy and the immediate

postpartum period. 13) Histopathology of HG shows

subepidermal blisters with eosinophil infiltration.

Bullous pemphigoid (BP)

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.

Histopathology Direct immunofluorescence

Subepidermal blister formation. Linear deposition of IgG along BMZ.

Skin lesion

Pemphigoid

Staphylococcal scalded skin syndrome (SSSS) and bullous impetigo are blistering diseases caused by exotoxins (exfoliative

toxin, ET) produced by Staphylococcus aureus. SSSS is common in newborns, infants, and patients with renal failure or

immunodeficiency. It is considered that ET disseminated through the bloodstream causes erythema and blisters throughout

the body. Nikolsky’s sign is also detected. In bullous impetigo, however, ET produces the blisters locally at the infected area.

The mechanisms of blister formation in both diseases had remained unclear until recently, although classic studies showed that

ET could induce blisters in neonatal mice in the 1970’s.

SSSS and pemphigus foliaceus (PF) share many similar features; (1) only the skin is affected, but not the mucous membrane,

(2) the blister formation occurs in the upper epidermis, (3) no necrotic keratinocytes precede the blisters, and (4) injections

of ET into neonatal mice induce superficial blisters whose histology is identical to PF. In addition, various experiments

indicated that several ET subtypes (exfoliative toxin A, B, and D) induced the blister formation by a cleavage of Dsg1 at Gul381

through their serine protease activity, while they do not cleave Dsg3. This reflects the histological manifestation in SSSS, of

which the lesion is localized in the skin. It is clinically significant that the inactivation of Dsg1 causes superficial skin blisters in

two different skin diseases, PF and SSSS. 23, 24)


Anti-BP180 autoantibodies in patients with BP are

generally considered to be pathogenic. In vitro studies

indicate that BP IgG activates the classical complement

pathway, thereby causing activation of inflammatory cells

with the degranulation and resulting dermal-epidermal

separation. 19) However, some reports suggested the

non-inflammatory mechanism of the blister formation;

anti-BP180 antibodies cause conformational changes

of BP180, to reduce functional BP180 molecules. 20) The

autoantibodies from most patients with BP recognize the

NC16a domain, which is a non-collageneous region of

BP180, located just outside the cell membrane.

In contrast, anti-BP230 antibodies may not directly

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

are detected only in some patients with BP. 21)

Autoantibodies against type VII collagen are associated

with dysfunction of anchoring fibrils in dermal-epidermal

junctions. Direct immunofluorescence staining shows

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

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-

chains (290 kDa). Each chain consists of a 145 kDa

non-collagen (NC1) domain, a typical collageneous

domain, and a 34 kDa non-collagen (NC2) domain.

Epitopes recognized by autoantibodies from EBA

patients are mainly on NC1. NC2 is also considered to

contain minor epitopes. 22)

Bullous pemphigoid (BP) and anti-BP180 and anti-BP230 IgG autoantibodies

Epidermolysis bullosa acquisita (EBA) and anti-type VII collagen IgG autoantibodies

EBA

BP

Epidermal side

Dermal side

Epidermal side

Dermal side

ComplementNC16a

Blister

Anti-BP230

Anti-BP180

Anchoring fibril

BP180

BP230

Anchoring fibril

BP180

BP230

Anti-type VII collagen

Blister

Topic

Sites of the skin blister formation in BP and EBA

ET: Exfoliative toxin

Dsg1

ET

- Desmoglein 1 is a target in bullous infectious diseases -

Profile Kit Type VII collagenDSG 1

DSG 3

BP180

BP230

1 2 3 4 5 6 7 8 9 10 11 12ABCDEFGH

Calibrator 1 (0 U/mL)Calibrator 2 (100 U/mL)Dsg1Dsg3

A:B:C:D:

BP180BP230Type VII collagenAssay control (Human IgG)

E:F:G:H:

Detects 5 anti-skin antibodiesin a single assay Pemphigus Bullous pemphigoid EBA

epidermis side

dermis side

epidermis side

dermis side

Code No. Product Volume Storage Antigens / Description

RG-M7593-D

RG-M7612-D

RG-M7613-D

RG-7845R2

RG-7115R

48 wells

48 wells

48 wells

48 wells

8 wells x 12

2-8 °C

2-8 °C

2-8 °C

2-8 °C

2-8 °C

MESACUP Dsg1 & Dsg3 ELISA TEST SYSTEM

MESACUP BP180 TEST

MESACUP BP230 TEST

Anti-Type VII collagen ELISA Kit (For Research use only)

Anti-Skin Profile ELISA Kit (For Research use only)

Recombinant human Dsg1 and Dsg3

Recombinant human BP180 NC16a

Recombinant human BP230 (NT and CT)

Recombinant human type VII collagen NC1 and NC2

Recombinant human Dsg1, Dsg3, BP180, BP230 and type VII collagen

Code No. Product Volume Storage Antigens / Description

RG-7880EC-D

RG-7885EC-D

RG-7695EC-D

RG-7613EC-D

RG-7845E

RG-7115EC-D

48 wells

48 wells

48 wells

48 wells

48 wells

8 wells x 12

2-8 °C

2-8 °C

2-8 °C

2-8 °C

2-8 °C

2-8 °C

MESACUP-2 TEST Desmoglein 1

MESACUP-2 TEST Desmoglein 3

MESACUP BP180 TEST

MESACUP BP230 TEST

MESACUP Anti-Type VII collagen TEST

MESACUP Anti-Skin Profile TEST

Recombinant human Dsg1

Recombinant human Dsg3

Recombinant human BP180 NC16a

Recombinant human BP230 (NT and CT)

Recombinant human type VII collagen NC1 and NC2

Recombinant human Dsg1, Dsg3, BP180, BP230 and type VII collagen

For US customers

For EU customers

Value was determined by analysis with 30 PV, 30 PF, 60 BP, 30 EBA and 42 normal samples.

ELISA Index (U/mL)

Negative Positive

< 15 ≥ 15MESACUP Anti-Skin Profile TEST*

Concordance Disconcordance Concordance Rate (%)

Dsg1

Dsg3

BP180

BP230

Type VII collagen

189

191

191

189

189

3

1

1

3

3

98.4

99.5

99.5

98.4

98.4

Types of pemphigus Anti-Dsg3 antibody Anti-Dsg1 antibody

Mucosal-dominant PV

Mucocutaneous PV

PF

Positive

Positive

Negative

Negative

Positive

Positive

ELISA Index (U/mL)

Positive

≥ 20MESACUP-2 TEST Desmoglein 1†

MESACUP Dsg1 & Dsg3 ELISA TEST SYSTEM†† ≥ 20

MESACUP-2 TEST Desmoglein 3† ≥ 20

*U.S. Customers : for research use only (Not for use in diagnostic procedures)

† For EU Customers only†† For U.S. Customers only



Differential diagnosis of pemphigus

Screening of autoimmune blistering diseases

Immunofluorescence assay may reveal differences in

staining patterns between PV and PF according to the

tissue distribution of Dsg1 and 3 (see page 4). However,

the immunofluorescence patterns of tissues are difficult

to clearly discriminate between the two diseases.

MESACUP Desmoglein ELISA kits are for measurements

of anti-Dsg1 and anti-Dsg3 antibodies in serum, using the

recombinant Dsg1 or Dsg3 as the solid-phase antigen.

These ELISA kits provide sensitive and specific assays

for the differential serological diagnosis between PV and

PF on the basis of their characteristic profiles of auto

antibodies. 25)

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

serum. These anti-skin autoantibodies can be detected

simultaneously in a single assay.

Interpretation of results with MESACUP Desmoglein ELISA kits

Disease classification of pemphigus by anti-Dsg antibody profiling

Presence of anti-Dsg1 and anti-Dsg3 antibodies in PV, PF, BP, and normal sera

Interpretation of results with MESACUP Anti-Skin Profile TEST*

Comparison of existing individual ELISA kits for autoimmuneblistering diseases and MESACUP Anti-Skin Profile TEST*

ELISA kits for the diagnosis of autoimmune blistering diseases

0

50

100

150

200

250Anti-Dsg1

PV(27/39)

PF(31/31)

BP(0/45)

Normal(0/180)

ELI

SA

Ind

ex

Anti-Dsg3

PV(39/39)

PF(0/31)

BP(1/45)

Normal(0/180)

20 20

ELISA Index (U/mL)

Negative Positive

< 9 ≥ 9

< 9

MESACUP BP180 TEST *1

MESACUP BP230 TEST *2 ≥ 9

Active stage Remission Total

MESACUP BP180 TEST

MESACUP BP230 TEST

MESACUP BP180 TEST +

MESACUP BP230 TEST

84% （54 / 64）

58% （37 / 64）

94% （60 / 64）

65% （113 / 175）

78% （136 / 175）

98% （172 / 175）

70% （167 / 239）

72% （173 / 239）

97% （232 / 239）

*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.

†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

Values were determined by ROC analysis with 49 EBA, 20 PF, 20 BP and 266 normal samples.

ELISA Index (U/mL)

Negative Positive

< 6 ≥ 6MESACUP Anti-Type VII collagen TEST

Interpretation of results with MESACUP Anti-Type VII collagen TEST*

Presence of anti-type VII collagen antibodies in EBA, PV, BP, and normal sera ‡

*U.S. Customers : for research use only (Not for use in diagnostic procedures)‡Data for EU Customers only.*U.S. Customers : for research use only (Not for use in diagnostic procedures)

12 Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid - 13Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid -12 Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid - 13Autoimmune Blistering Diseases - Diagnostic Methodology for Pemphigus and Pemphigoid -

Monitoring disease activities by ELISA

Clinical data using MESACUP Desmoglein kits suggest

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).

In recent studies, the amount of anti-Dsg1 and anti-Dsg3

antibodies decreased significantly after rituximab and

intravenous immunoglobulin (IVIG) therapies. 29-31)

Monitoring disease activity in mucocutaneous PV with MESACUP Desmoglein ELISA kits

600

400

200

0

5040302010

0

Anti-

desm

ogle

in a

ntib

odie

s (U

/mL)

PDAI

0 14 37

Days after treatment

Anti-Dsg 3

Anti-Dsg 1

* PDAI: Pemphigus Disease Area Index


Ant

i-typ

e VI

I col

lage

n an

tibod

ies

(U/m

L)


6000

4000

2000

027 75 56 0

The amount of anti-type VII collagen antibodies decreased

with the improvement of cutaneous symptoms in a patient

with EBA after treatment. 28, 36, 37)

Monitoring disease activity in EBA with MESACUP Anti-Type VII collagen TEST*

Differential diagnosis of epidermolysis bullosa acquisita (EBA)

Differential diagnosis of BP

MESACUP Anti-Type VII collagen TEST is an ELISA kit

using the recombinant NC1 and NC2 domains of type VII

collagen for measurement of anti-type VII collagen

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)‡

MESACUP BP180 TEST is an ELISA kit for the detection

of anti-BP180 antibodies in human serum, using the

recombinant BP180 NC16a protein as the immobilized

antigen. The sensitivity of the kit is 84% (54/64) in patients

with BP in the active stage. 26)

MESACUP BP230 TEST is an ELISA kit using both

the recombinant N-terminal and C-terminal domains

of BP230 for detection of anti-BP230 antibodies in

human serum. The sensitivity is 58% (37/64) in patients

diagnosed with BP in the active stage. Most significantly,

higher clinical sensitivity (97%) can be achieved by

combining the results of both BP180 and BP230 ELISA

kits. 27)

Interpretation of results with MESACUP BP180 and MESACUP BP230

Clinical sensitivity of anti-BP180 and anti-BP230 ELISA for BP sera†


ELI

SA

Ind

ex

Anti-BP230

BP(Active stage)

(37/64)

BP(Remission)(135/175)

PF/PV(1/94)

Normal(0/109)

0

50

100

150

200

250Anti-BP180

BP(Active stage)

(54/64)

BP(Remission)(113/175)

PV/PF(1/94)

Normal(5/336)

9 9

ELI

SA

Ind

ex

0

100

200

300

400

EBA(47/49)

PV(0/20)

BP(0/20)

Normal(1/266)

6

Presence of anti-BP180 and anti-BP230 antibodies in BP, PV/PF, and normal sera

Clinical manifestation of PV disease and anti-desmoglein antibody levels

Several clinical studies suggested that the data using

MESACAP BP180 TEST demonstrated that the

disappearance of blisters correlated with decrease in the

amount of anti-BP180 antibody. 27,33,34)

Additionally, in a previous report on one patient with

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

peripartum period. 35) These findings strongly suggest

the clinical importance of periodic measurements of the

autoantibodies in patients with BP.

Monitoring disease activity in BP with MESACUP BP180 TEST

Anti-

BP18

0 an

tibod

ies

(U/m

L)


1000

800

600

400

200

00 12 3528

Clinical manifestation of BP disease and anti-BP180 antibody levels

Clinical manifestation of EBA disease and anti-type VII collagen antibody levels

We are grateful to Drs. Masayuki Amagai and Jun Yamagami of Keio University School of Medicine for data and valuableadvice, and to Dr. Ken Ishii of Teikyo University Chiba Medical Center and Dr. Takashi Hashimoto of Kurume University School of Medicine for data.

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References

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