ag serological diagnosis of autoimmune … against...euroimmun medizinische labordiagnostika ag...

EUROIMMUNM e d i z i n i s c h eL a b o r d i a g n o s t i k aA G

Serological diagnosis of autoimmune dermatoses: Dermatology Mosaic 9 (Order number: FA 1501-9)

EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Telephone +49 451 58 550 · Fax 5855591 · e-mail [email protected] · www.euroimmun.de

Desmoglein 1 BP230 whole length

Oesophagus

Desmoglein 3

BP230 gC Oesophagus

Desmoglein 1

Desmoglein 3

BP230 gC

BP230 whole length

BP180 (NC16A-4X)BP180 (NC16A-4X)

epid. BM pos. pos. Desmos. pos. neg.

neg.

neg.

pos.

pos.

pos. neg.

neg.neg.

Bullous pemphigoid (anti-epid. basement membrane pos.) Pemphigus foliaceus (anti-desmoglein 1 pos.)

FA_1501_I_UK_A01, 10/2009

Introduction

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune intraepider-mal blistering skin diseases characterized by autoantibodies to desmoglein 1 (Dsg1) and desmoglein 3 (Dsg3), respectively. An-tigen-specifi c assays using the immuno-genic extracellular Dsg domains can be ap-plied for serological distinction between PV and PF. In this study, two ELISA based on human cell-expressed Dsg1 and Dsg3 were assessed for the fi rst time with respect to their diagnostic performance and their suit-ability for therapy monitoring.

Methods

Ectodomains of Dsg1 and Dsg3 were ex-pressed in HEK293 cells, purifi ed from cell culture supernatant, and used as solid phase in ELISA for autoantibody determination of circulating IgG. Assay evaluation was per-formed with a panel of sera from 121 clini-cally characterized patients with pemphigus (71 PV, 50 PF), 48 with bullous pemphigoid (BP), 21 with linear IgA dermatosis (LAD) and 401 healthy blood donors (HBD).

Results

Autoantibodies to Dsg1 were detected in 48 (96%) PF sera and in 0.9% of control

subjects (1 BP, 0 LAD, 3 HBD). Reactivity to Dsg3 was found in all (100%) PV sera and in 0.4% of the control cohort (1 BP, 0 LAD, 1 HBD). Thus, the applied test systems revealed a sensitivity of 96% and 100% for anti-Dsg1 and anti-Dsg3, respectively, at a specifi city of more than 99%. Levels of circulating autoantibodies, which were measured in 12 patients, correlated with the clinical severity in all cases.

Conclusion

ELISA based on human cell-expressed ectodomains of Dsg1 or Dsg3 represent highly sensitive and specifi c test systems, suitable for both, the serological diagnosis of patients with pemphigus and the moni-toring of the disease activity.

EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 451 58550 · Fax 5855591 · E-mail [email protected]

ELISA using ectodomains of desmoglein 1 and 3 expressed in HEK293 for sensitive and specifi c detection of

pemphigus autoantibodies

C. Daehnrich1, A. Rosemann1, C. Probst1, L. Komorowski1, W. Schlumberger1,

W. Stoecker1, A. Recke2, C. Rose2, D. Zillikens2, E. Schmidt2

1Institute for Experimental Immunology, affi liated to EUROIMMUN AG, Luebeck, Germany2Department of Dermatology, University of Luebeck, Germany

Desmoglein 1 and 3: Constituents of desmosomes Different patient cohorts in the Anti-Desmoglein 1 ELISA. Different patient cohorts in the Anti-Desmoglein 3 ELISA.

Panel n anti-Dsg1 positive anti-Dsg3 positive

Pemphigus vulgaris 71 33 (47%) 71 (100%)

Pemphigus foliaceus 50 48 (96%) 0

Linear IgA dermatosis 21 0 0

Bullous pemphigoid 48 1 (2%) 1 (2%)

Blood donors 401 3 (1%) 1 (0.2%)

Specifi city 470 99.1% 99.6%

PF PV BP LAD HBD

An

ti-D

sg1

ELI

SA

(O

D)

0

1

2

3

4

(48/50) (1/48) (0/21) (3/401)(33/71)

0.5

PV PF BP LAD HBD

An

ti-D

sg3

ELI

SA

(O

D)

0

0.5

2

3

4

(71/71) (1/48) (0/21) (1/401)(0/50)

1

One pemphigus foliaceus patient

Weeks

7040 147 161

An

ti-d

esm

og

lein

1 a

nti

bo

dy

tite

r (R

U/m

l)

0

200

400

600

800

1000

1200

1400

1600

1800

Dis

ease

act

ivit

y sc

ore

0

1

2

3

4

5

14620 320

20

40

60

80

100

0

1

2

3

4

5

One pemphigus vulgaris patientA

nti

-des

mo

gle

in 3

an

tib

od

y ti

ter

(RU

/ml)

Dis

ease

act

ivit

y sc

ore

Weeks

Scientifi c presentation at the 9th Dresden Symposium on Autoantibodies (Dresden, Germany, September 2009)

Basal keratino-cytes

Lamina lucida

Lamina densa

Sublamina densa

Dsg1

Dsg3

Dsc1

Introduction

Bullous pemphigoid (BP) and pemphigoid gestationis (PG) are acquired autoimmune subepidermal blistering diseases char-acterized by autoantibodies against the hemidesmosomal proteins BP180/type XVII collagen and BP230. The vast majority of BP and PG patients demonstrate autoanti-body binding to epitopes clustered within the 16th noncollagenous domain NC16A of BP180 (a).

Methods

In order to achieve effi cient protein expres-sion in E. coli, four copies of the NC16A domain were fused to a carboxyterminal polyhistidine tag (b). This strategy provides a high yield by simple purifi cation of the target protein under denaturing conditions, without the need to cleave off a heterologous fusion partner. The protein, purifi ed by metal chelate affi nity chromatography (c), migrated consist-ent to its calculated mass of 36.6 kDa when separated by SDS-PAGE (lane 2). A mono-clonal antibody specifi c for hexahistidin (lane 3) and serum from a patient with BP (lane 4) but not from a healthy blood donor (lane 5) recognized this recombinant form of NC16A by immunoblot analysis. Based on this tetra-meric protein as antigenic substrate an ELISA for the detection of autoantibodies against BP180 was developed and evaluated.

Results

Autoantibodies against BP180 were found in 106 (89.8%) of 118 randomly selected BP sera and in all of 20 (100%) randomly selected PG sera, whereas only 2.1% of a large cohort of control subjects were posi-tive in this assay, including patients with rheumatoid arthritis (RA; 2 of 107), progres-sive systemic sclerosis (PSS; 2 of 50), sys-temic lupus erythematosus (SLE; 1 of 72), and healthy blood donors (not shown, 10 of 494). Levels of circulating autoantibod-ies against BP180 (see columns) paralleled disease activity (see line graph) in pemphi-goid patients.

Discussion

The new ELISA showed better perform-ance than formerly described test systems. We propose that the multimer form of the

autoantigen not only increases immunore-activity, but also accessibility of the target epitope which facilitates binding of autoan-tibodies. A second advantage of using the recombinant BP180 as target antigen is the clear characterisation of the autoanti-body specifi city. In conclusion, the use of tetrameric NC16A in ELISA signifi cantly improves making the diagnosis and moni-toring disease activity of patients with BP and PG.

EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 451 58550 · Fax 5855591 · E-mail [email protected]

Sensitive and specifi c detection of pemphigoid autoantibodies by an Enzyme-linked immunosorbent assay using multimers

of the NC16A domain of BP180 as antigenC. Probst1, C. Daehnrich1, L. Komorowski1, A. Rosemann1, E. Schmidt2, W. Schlumberger1, C. Sitaru3 C. Rose3, W. Stoecker1, and D. Zillikens3

1Institute of Experimental Immunology, affi liated to EUROIMMUN, Luebeck, Germany2Department of Dermatology, University of Wuerzburg, Germany

3Department of Dermatology, University of Luebeck, Germany

Panel nTetrameric

NC16A ELISA

GST-NC16A

ELISA

BP 118 106 (89.8%) 105 (89.0%)

PG 20 20 (100.0%) 20 (100.0%)

RA 107 2 (1.9%) 5 (4.7%)

PSS 50 2 (4.0%) 4 (8.0%)

SLE 72 1 (1.4%) 3 (4.3%)

Sensitivity 118 89.8% 89.0%

Specifi city 229 97.8% 94.8%

BP(106/118)

EU

RO

IMM

UN

(R

U/m

l)

0,1

1

10

100

1000

10000

RA(2/107)

PSS(2/50)

SLE(1/72)

PG(20/20)

Basal keratinocyte

NH2

COOH

NC16A

C15

6 x HisNC16A NC16A NC16A NC16A

pET24d-N-(NC16A)4

6145 bp

Kan

lacI

T7His-(NC16A

)4

a) Schematic illustration of BP180 structure

b) Expression construct and recombinant NC16A-4X used

c) Initial validation of purifi ed recombinant protein

Weeks

0 8 10 14 16 20 24 28 32 36 40 48 52 56 60 66

RU

/ml

0

20

40

60

80

100

120

140

160

180

200

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Dis

ease

act

ivit

y sc

ore

Scientifi c presentation at the 8th Dresden Symposium on Autoantibodies (Dresden, Germany, September 2007)

2 kDa3 kDa

6 kDa

14 kDa

21 kDa

31 kDa36 kDa

55 kDa66 kDa

97 kDa116 kDa

185 kDa

1 2 3 4 5

EA_1502_I_UK_A01, 10/2007


DSG1

DSG1

DSG1

DSG1

DSG1

DSG1

DSG1

DSG1

EUROIMMUN Microplate ELISA

Autoantibody determination:AMA M2-3E (IgG)ANCA Profile (IgG)ANA Screen (IgG)ANA Screen 9 or 11 (IgG)ANA VarioProfile (IgG)BP180-NC16A-4X (IgG)BP230-CF (IgG)C1q (IgG)cardiolipin (IgA, IgG, IgM, IgAGM)circulating immune complexes (CIC)cyclic citrullinated peptide (CCP; IgG)centromere protein B (IgG)desmoglein 1 (IgG)desmoglein 3 (IgG)double-stranded DNA (dsDNA, nDNA; IgG)dsDNA-NcX (IgG)ENA Pool (IgG)ENA PoolPlus (IgG)ENA ProfilePlus 1 or 2 (IgG)ENA SLE Profile 1 or 2 (IgG)GADGAD/IA-2 Poolglomerular basement membrane (GBM; IgG)ß2-glycoprotein 1 (IgA, IgG, IgM, IgAGM)histones (IgG)IA-2intrinsic factor (IgG)Jo-1 (IgG)liver cytosolic antigen type 1 (LC-1; IgG)liver-kidney microsomes (LKM-1; IgG)myeloperoxidase (MPO; IgG)nRNP/Sm (IgG)nucleosomes (IgG)p53 (IgG)parietal cells (PCA; IgG)PM-Scl (PM-1; IgG)phosphatidylserine (IgA, IgG, IgM, IgAGM)proteinase 3 (IgG)PR3 hn-hr (IgG)PR3 capture (IgG)rheumatoid factor (IgA, IgG, IgM)ribosomal P-proteins (IgG)Sa (IgG)Scl-70 (IgG)single-stranded DNA (ssDNA; IgG)SLA/LP (IgG)Sm (IgG)SS-A (Ro; IgG)SS-B (La; IgG)thyroglobulin (TG; IgG)thyroid peroxidase (TPO; IgG)tissue transglutaminase (endomy.; IgA, IgG)TSH receptor (TBII; IgG)TRAk Fast (IgG)

Further autoimmune diagnostics:gliadin (GAF-3X; IgA, IgG)Saccharomyces cerevisiae (IgA, IgG)

Infectious serology:Adenovirus (IgA, IgG, IgM)Borrelia (IgG, IgM)Borrelia VlsE (IgG)Chlamydia pneumoniae (IgA, IgG, IgM)Chlamydia trachomatis (IgA, IgG, IgM)Cytomegalovirus (IgG, IgM)Diphtheria toxoid (IgG)Epstein-Barr virus capsid ag (IgA, IgG, IgM)Epstein-Barr virus early ag (IgA, IgG, IgM)Epstein-Barr virus nuclear ag, EBNA-1 (IgG)Helicobacter pylori (IgA, IgG)Helicobacter pylori CagA (IgA, IgG)HSV-1 (glycoprotein C1; IgA, IgG, IgM)HSV-2 (glycoprotein G2; IgA, IgG, IgM)HSV-1/2 Pool (IgA, IgG, IgM)Influenza virus type A (IgA, IgG, IgM)Influenza virus type B (IgA, IgG, IgM)Legionella pneumophila (IgA, IgG, IgM)Measles virus (IgG, IgM)Mumps virus (IgG, IgM)Mycoplasma pneumoniae (IgA, IgG, IgM)Parainfluenza virus Pool (IgA, IgG, IgM)RSV (IgA, IgG, IgM)Rubella virus (IgG, IgM)SARS-CoV (IgG)TBE virus (IgG, IgM)Tetanus toxoid (IgG)Toxoplasma gondii (IgG, IgM)Treponema pallidum (IgG, IgM)Varicella zoster virus (IgG, IgM)Yersinia enterocol. virulence fact. (IgA, IgG)

Allergology:total IgEAllercoat™ 6-ELISA (600 differentallergens and allergen mixtures)

Serum proteins and tumour markers:anti-p53

* Currently not available as IVD in the EU.

Made in Germany

Anti-Desmoglein 1 ELISA (IgG)

Indication: Test system for the in vitro determination of antibodies against desmoglein 1 in human serum or plasma for the diagnosis of the following diseases: pemphigus foliaceus, pemphigus vulgaris.

Clinical significance: Autoantibodies against desmoglein 1 and 3 are markers for pemphigus diseases, which can be clinically and immunopathologically subdivided into 4 different forms: pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus and IgA pemphigus.

Pemphigus vulgaris always affects the mucous membranes. The majority of patients initially only develop lesions in the mucosa of the mouth. In the course of the disease some patients show flaccid blisters at the integument, particularly on parts of the body which are exposed to pressure and friction. Patients with pemphigus vulgaris who show damage exclusively to the mouth mucosa exhibit IgG antibodies only against desmoglein 3, whereas patients with lesions of the skin and mucosa produce antibodies against desmoglein 1 and 3. In patients with pemphigus foliaceus, blisters are rarely found due to a very superficial cleft formation in the stratum granulosum. The disease is rather characterised by scaly crusts, especially in the seborrheic areals. The mucosa is never affected. Congruently, pemphigus foliaceus is only associated with desmoglein 1. Paraneoplastic pem-phigus is always associated with neoplasia. The resulting immune response is not only directed against desmoglein 3 but also against other proteins of desmosomal plaques such as envoplakin, periplakin, desmoplakin and plektin. IgA pemphigus is characterised by IgA antibodies against desmoglein 1 and desmocolin 1. Desmoglein 1 and 3 are cadherins, which are calcium-dependent transmembrane glycoproteins of epidermal desmosomes. They are com-ponents of the maculae adherentes and permit the cell-to-cell contact in the epidermis and the surface mucosa via homophilic and heterophilic extracellular binding. The pathogenetic relevance of autoantibodies against desmo-glein 1 and 3 is well proven. For example, the injection of serum from pemphigus patients into neonatal mice leads to blister formation. The exact mechanisms which cause the blister formation, however, are unknown. Clinical sensitivity and specificity: Sera from 50 patients with pemphigus foliaceus, 71 patients with pemphigus vulgaris, a control panel of 69 patients with other autoimmune diseases and 401 healthy blood donors were investigated using the EUROIMMUN Anti-Desmoglein 1 ELISA. The sensitivity of the ELISA for pemphigus foliaceus was 96.0%, with a specificity of 99.1%. In the pemphigus vulgaris panel 46.5% of patients were found positive.

Application of the Anti-Desmoglein 1 ELISA: In the diagnosis of pemphigus, determination of circulating autoantibodies using indirect immunofluorescence (on primate oesophagus as sensitive substrate) has proven successful. However, it does not allow differentiation between antibodies against desmoglein 1 and desmoglein 3. ELISA using desmoglein 1 and 3 offers the same sensitivity and specificity as IIFT. In most cases the Anti-Desmoglein 1 ELISA and Anti-Desmoglein 3 ELISA are sufficient to diagnose pemphigus. In suspected pemphigus cases with a negative ELISA result IIFT should be carried out in addition. The Anti-Desmoglein 3 ELISA is of particular importance in lesions of the mouth mucosa to differentiate pemphigus vulgaris from Lichen ruber mucosae, benign aphtha, Behcet‘s disease and Steven-Johnson syndrome.

The Anti-Desmoglein 1 ELISA and Anti-Desmoglein 3 ELISA are highly sensitive and specific test systems for the diagnosis of pemphigus diseases. In untreated patients a positive result in the Anti-Desmoglein 3 ELISA alone suggests the presence of pemphigus vulgaris with only mucosa involvement. If both the Anti-Desmoglein 3 ELISA and the Anti-Desmoglein 1 ELISA are positive, this indicates pemphigus vulgaris with mucosa and skin involvement. A positive Anti-Desmoglein 1 ELISA result alone is indicative of pemphigus foliaceus. The antibody levels of desmoglein 1 and 3 in the serum generally correlate with the severity and activity of the disease and the therapy success.

Panel nAnti-

Desmoglein 1positive

Pemphigus foliaceus (PF)

50 48 (96.0 %)

Pemphigusvulgaris (PV)

71 33 (46.5 %)

Asymptomatic blood donors (BD)

401 3 (0.7 %)

Bullous pemphigoid (BP)

48 1 (2.1 %)

Linear IgAdermatosis (LAD)

21 0 (0.0 %)

Sensitivity forpemphigus foliaceus

50 48 (96.0 %)

Specificity for pemphigus foliaceus

470 4 (99.1 %)

EUROIMMUN AG · 23560 Luebeck (Germany) · Seekamp 31 · Telephone +49 451 58550 · Fax 5855591 · E-mail [email protected]


Linearity: The linearity of the ELISA was determined by assaying 4 serial dilutions of 6 serum samples. The linear regression was calculated, R2 amounting to > 0.95 in all samples. The Anti-Desmoglein 1 ELISA (IgG) is linear in at least the tested concentration range (9-197 RU/ml).

Reproducibility: The reproducibility of the test was investiga-ted by determining the intra- and inter-assay coefficients of variation using 3 sera. The intra-assay CVs are based on 20 determinations and the inter-assay CVs on 4 determinations performed in 6 different test runs.

Reference range: Levels of anti-desmoglein 1 antibodies were determined in 401 sera from healthy blood donors of between 18 and 68 years of age (151 women, 250 men) using the EURO-IMMUN ELISA. The mean concentration of antibodies against desmoglein 1 was 1.8 RU/ml and the values ranged from 0.4 to 39.0 RU/ml. With a cut-off of 20 RU/ml, 0.7% of the blood donors were anti-desmoglein 1 positive.

ROC analysis: In an analysis of 50 samples from patients with pemphigus foliaceus and 470 control samples the following results were achieved:

Correlation of the EUROIMMUN and MBL Anti-Desmoglein 1 ELISAs: The antibody concentration was determined in 50 sera from patients with pemphigus foliaceus using the Anti-Desmoglein 1 ELISAs from EUROIMMUN and MBL. The qua-litative results of the ELISAs were 94% in agreement.

The antibody concentration was measured in 69 patients with other autoimmune diseases (bullous pemphigoid, linear IgA dermatosis) using the Anti-Desmoglein 1 ELISAs from EURO-IMMUN and MBL. The qualitative results of the ELISAs were 97% in agreement.

Technical data:

Antigen Recombinant, expression in mammalian cells, extracellular domain of desmolein 1 (5 subdomains).

Calibration Quantitative, in relative units per milliliter (RU/ml). Calibration serum 1: 200 RU/ml Calibration serum 2: 20 RU/ml ; cut-off Calibration serum 3: 2 RU/ml

Sample dilution Serum or plasma; 1 : 101 in sample buffer.

Reagents Ready for use, with the exception of the wash buffer (10x). Colour-co-ded solutions, in most cases exchangeable with those in other EURO-IMMUN ELISA kits.

Test procedure 30 min / 30 min / 15 min. Room temperature. Fully automatable.

Measurement 450 nm. Reference wavelength between 620 nm and 650 nm.

Test kit format 48 break-off wells. Kit includes all necessary reagents.

Order no. EA 1495-4801 G

Test characteristics Anti-Desmoglein 1 ELISA (IgG)

Intra-assayvariation, n = 20

Inter-assayvariation, n = 4 x 6

SerumMean value

(RU/ml)CV (%)

Mean value (RU/ml)

CV (%)

1 47 4.0 46 3.7

2 73 3.1 70 6.1

3 111 3.3 114 6.0

Blood donors, n = 401

Percentile 99th 100th

Cut-off 13.4 RU/ml 39.0 RU/mlPF PV BP LAD BD

Ant

i-Dsg

1 EL

ISA

(OD

)

0

1

2

3

4

(48/50) (1/48) (0/21) (3/401)(33/71)

0.5

Cut-off Specificity Sensitivity

16.7 RU/ml 98.9 % 96.0 %

21.1 RU/ml 99.2 % 96.0 %

RU/ml0.1 1 10 100 1000

Freq

uen

cyn

0

20

40

60

80

100

120

401 Blood donors

PF patients,

n = 50

EUROIMMUN

positive negative

MBLpositive 47 2

negative/borderline

1 0

Controls,

n = 69

EUROIMMUN

positive negative

MBLpositive 0 1

negative/borderline

1 67

EUROIMMUN Immunoblots

Autoantibody determination:

EUROASSAY:flexible profiles of up to 7 antigens from:ENA and related antigens: nRNP/Sm, Sm, SS-A, Ro-52, SS-B, Scl-70, Jo-1,dsDNA, histones, nucleosomes, CENP B,PM-Scl, ribosomal P-proteins, AMA M2liver antigens: LKM-1, LC-1, SLA/LP,AMA M2, M4, M9ANCA antigens: MPO, PR3thyroid antigens: TG, TPO

EUROLINE:ANA Profile 1: nRNP/Sm, Sm, SS-A, Ro-52,SS-B, Scl-70, Jo-1, CENP B, dsDNA,nucleosomes, histones, ribosomal P-proteinsANA Profile 3: nRNP/Sm, Sm, SS-A, Ro-52,SS-B, Scl-70, PM-Scl, Jo-1, CENP B, PCNA, dsDNA, nucleosomes, histones, ribosomal P-proteins, AMA M2Anti-ENA Profile 1: nRNP/Sm, Sm, SS-A, Ro-52,SS-B, Scl-70, Jo-1Myositis Profile: Mi-2, Ku, PM-Scl,Jo-1, PL-7, PL-12, Ro-52Liver Profiles: AMA M2, 3E (BPO), Sp100, PML,gp210, LKM-1, LC-1, SLA/LP, Ro-52Neuronal Antigens Profile 2: amphiphysin, CV2.1**PNMA2 (Ma-2/Ta), Ri, Yo, HuAnti-Ganglioside Profile 1: GM1, GD1b, GQ1bAnti-Ganglioside Profile 2: GM1, GM2, GM3, GD1a, GD1b, GT1b, GQ1bANCA Profiles: MPO, PR3, GBM

EUROLINE-WB:neuronal antigens (+ recomb. Hu, Yo, Ri)HEp-2 cell antigens (+ SS-A and Ro-52, CENP B)

Infectious serology:

EUROLINE:Bordetella pertussis (IgA, IgG)Borrelia-RN-AT (p18, p19, p20, p21, p58, OspC,p39, p83, LBb, LBa, VlsE Bg, VlsE Bb, VlsE Ba)EBV Profile (IgG, IgM, VCA gp125, VCA p19and EBNA-1, p22, EA-D)Hanta virus (IgG, IgM)Rubella virus (IgG)TORCH Profile* (T. gond., rubella, CMV, HSV-1, -2)

Westernblot:Borrelia burgdorferi (IgG, IgM)Borrelia afzelii (IgG, IgM)Borrelia garinii (IgG, IgM)Epstein-Barr virus (IgG, IgM)Helicobacter pylori (IgA, IgG)Treponema pallidum (IgG, IgM)Yersinia enterocol. virulence fact. (IgA, IgG)

EUROLINE-WB:

Anti-Borrelia (B. afzelii + rec. VlsE)Anti-HSV (HSV-1 + HSV-2 gG2)Treponema pallidum + cardiolipin

Allergology:

EUROASSAY: Domestic Animal Profile (IgE)Food Profile (IgE)Inhalation Profile (IgE)Insect Venom Profile (IgE)Latex Profile (IgE)Latex plus Profile (with ficus and fruit; IgE)

EUROLINE:Atopy Profile (IgE)Food Profile (IgE)Inhalation Profile (IgE)Paediatric Inhalation ProfilePollen–Food Cross Reaction Profile (IgE)

Software/Automation:

EUROLineScancamera system EUROBlotCamerascanner system EUROBlotScannerincubation processor EUROBlotMaster

EUROIMMUNRadioimmunoassays


thyroid peroxidase (TPO; IgG)thyroglobulin (TG; IgG)TSH receptor (IgG)acetylcholine receptor (ACHR; IgG)glutamic acid decarboxylase (GAD; IgG)insulin (IAA; IgG)P/Q calcium channel* (VGCC; IgG)tyrosine phosphatase (IA2; IgG)dsDNA (IgA/IgG/IgM)

Antigen determination:

thyroglobulin (TG)

Hormone determination:

free triiodothyronine (FT3)free thyroxine (FT4)thyrotropin (TSH)calcitonin

* Currently not available as IVD in the EU.** CV2 partial protein, which only contains the N-terminally localised epitopes of the antigen.

Made in Germany

Version: 07/09EA_1495_D_UK_A01



DSG3

DSG3

DSG3

DSG3

DSG3

DSG3

DSG3

DSG3








Made in Germany

Anti-Desmoglein 3 ELISA (IgG)

Indication: Test system for the in vitro determination of antibodies against desmoglein 3 in hu-man serum or plasma for the diagnosis of the following disease: pemphigus vulgaris.

Clinical significance: Autoantibodies against desmoglein 1 and 3 are markers for pemphigus diseases, which can be clinically and immunopathologically subdivided into 4 different forms: pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus and IgA pemphigus.

Pemphigus vulgaris always affects the mucous membranes. The majority of patients initially only develop lesions in the mucosa of the mouth. In the course of the disease some patients show flaccid blisters at the integument, particularly on parts of the body which are exposed to pressure and friction. Patients with pemphigus vulgaris who show damage exclusively to the mouth mucosa exhibit IgG antibodies only against desmoglein 3, whereas patients with lesions of the skin and mucosa produce antibodies against desmoglein 1 and 3. In patients with pemphigus foliaceus, blisters are rarely found due to a very superficial cleft formation in the stratum granulosum. The disease is rather characterised by scaly crusts, especially in the seborrheic areals. The mucosa is never affected. Congruently, pemphigus foliaceus is only as-sociated with desmoglein 1. Paraneoplastic pemphigus is always associated with neoplasia. The resulting immune response is not only directed against desmoglein 3 but also against other proteins of desmo-somal plaques such as envoplakin, periplakin, desmoplakin and plektin. IgA pemphigus is characterised by IgA antibod-ies against desmoglein 1 and desmocolin 1. Desmoglein 1 and 3 are cadherins, which are calcium-dependent trans-membrane glycoproteins of epidermal desmosomes. They are components of the maculae adherentes and permit the cell-to-cell contact in the epidermis and the surface mucosa via homophilic and heterophilic extracellular binding. The pathogenetic relevance of autoantibodies against desmo-glein 1 and 3 is well proven. For example, the injection of serum from pemphigus patients into neonatal mice leads to blister formation. The exact mechanisms which cause the blister formation, however, are unknown. Clinical sensitivity and specificity: Sera from 71 patients with pemphigus vulgaris, 50 patients with pemphigus foliaceus, a control panel of 69 patients with other autoimmune diseases and 401 healthy blood donors were investigated using the EUROIMMUN Anti-Desmoglein 3 ELISA. The sensitivity of the ELISA for pemphigus vulgaris was 100%, with a specificity of 99.6%. In the pemphigus foliaceus panel all patients were found negative.

Application of the Anti-Desmoglein 3 ELISA: In the diagnosis of pemphigus, determination of circulating autoantibodies using indirect immunofluorescence (on primate oesophagus as sensitive substrate) has proven successful. However, it does not allow differentiation between antibodies against desmoglein 1 and desmoglein 3. ELISA using desmoglein 1 and 3 offers the same sensitivity and specificity as IIFT. In most cases the Anti-Desmoglein 1 ELISA and Anti-Desmoglein 3 ELISA are sufficient to diagnose pemphigus. In suspected pemphigus cases with a negative ELISA result IIFT should be carried out in addition. The Anti-Desmoglein 3 ELISA is of particular importance in lesions of the mouth mucosa to differentiate pemphigus vulgaris from Lichen ruber mucosae, benign aphtha, Behcet‘s disease and Steven-Johnson syndrome.

The Anti-Desmoglein 1 ELISA and Anti-Desmoglein 3 ELISA are highly sensitive and specific test systems for the diagnosis of pemphigus diseases. In untreated patients a positive result in the Anti-Desmoglein 3 ELISA alone suggests the presence of pemphigus vulgaris with only mucosa involvement. If both the Anti-Desmoglein 3 ELISA and the Anti-Desmoglein 1 ELISA are positive, this indicates pemphigus vulgaris with mucosa and skin involvement. A positive Anti-Desmoglein 1 ELISA result alone is indicative of pemphigus foliaceus. The antibody levels of desmoglein 1 and 3 in the serum generally correlate with the severity and activity of the disease and the therapy success.

Panel nAnti-

Desmoglein 3positive

Pemphigus vulgaris (PV)

71 71 (100 %)

Pemphigusfoliaceus (PF)

50 0 (0 %)

Asymptomatic blood donors (BD)

401 1 (0.2 %)

Bullous pemphigoid (BP)

48 1 (2.1 %)

Linear IgAdermatosis (LAD)

21 0 (0,0 %)

Sensitivity forpemphigus vulgaris

71 71 (100 %)

Specificity for pemphigus vulgaris

470 2 (99.6 %)












EUROLINE-WB:


Allergology:









thyroglobulin (TG)




Made in Germany

Linearity: The linearity of the ELISA was determined by assaying 4 serial dilutions of 6 serum samples. The linear regression was calculated, R2 amounting to > 0.95 in all samples. The Anti-Desmoglein 3 ELISA (IgG) is linear in at least the tested concentration range (14-195 RU/ml).

Reproducibility: The reproducibility of the test was investiga-ted by determining the intra- and inter-assay coefficients of variation using 3 sera. The intra-assay CVs are based on 20 determinations and the inter-assay CVs on 4 determinations performed in 6 different test runs.

Reference range: Levels of anti-desmoglein 3 antibodies were determined in 401 sera from healthy blood donors of between 18 and 68 years of age (151 women, 250 men) using the EURO-IMMUN ELISA. The mean concentration of antibodies against desmoglein 3 was 1.8 RU/ml and the values ranged from 0.4 to 25.3 RU/ml. With a cut-off of 20 RU/ml, 0.2% of the blood donors were anti-desmoglein 3 positive.

ROC analysis: In an analysis of 71 samples from patients with pemphigus vulgaris and 470 control samples the following results were achieved:

Correlation of the EUROIMMUN and MBL Anti-Desmoglein 3 ELISAs: The antibody concentration was determined in 71 sera from patients with pemphigus vulgaris using the Anti-Desmoglein 3 ELISAs from EUROIMMUN and MBL. The qua-litative results of the ELISAs were 99% in agreement.

The antibody concentration was measured in 69 patients with other autoimmune diseases (bullous pemphigoid, linear IgA dermatosis) using the Anti-Desmoglein 3 ELISAs from EURO-IMMUN and MBL. The qualitative results of the ELISAs were 97% in agreement.

Technical data:

Antigen Recombinant, expression in mammalian cells, extracellular domain of desmoglein 3 (5 subdomains).



Reagents Ready for use, with the exception of the wash buffer (10x). Colour-co-ded solutions, in most cases exchangeable with those in other EURO-IMMUN ELISA kits.



Test kit format 48 break-off wells. Kit includes all necessary reagents.

Order no. EA 1496-4801 G

Test characteristics Anti-Desmoglein 3 ELISA (IgG)

Intra-assay variation, n = 20

Intra-assay variation, n = 4 x 6

SerumMean value

(RU/ml)CV (%)

Mean value (RU/ml)

CV (%)

1 42 4.4 44 6.1

2 63 2.6 69 5.3

3 155 5.9 163 3.3

RU/ml0.1 1 10 100 1000

Freq

uen

cyn

0

20

40

60

80

401 Blood donors

Blood donors, n = 401


Cut-off 11.4 RU/ml 25.3 RU/ml


15.9 RU/ml 99.4 % 100 %

21.8 RU/ml 99.6 % 100 %

PV PF BP LAD BD

Ant

i-Dsg

3 E

LIS

A (O

D)

0

0.5

2

3

4

(71/71) (1/48) (0/21) (1/401)(0/50)

1

PV patients, n = 71EUROIMMUN

positive negative

MBLpositive 70 0

negative/borderline

1 0

Controls,

n = 69

EUROIMMUN

positive negative

MBLpositive 0 1

negative/borderline

1 67



BP180

BP180

BP180

BP180

BP180

BP180

BP180

BP180

EUROIMMU AG · D-23560 Luebeck (Germany) · Seekamp 31 · Phone +49 451 58550 · Fax 5855591 · E-mail [email protected] · www.euroimmun.de








Made in Germany

Anti-BP180-NC16A-4X ELISA (IgG)

Indications: Test system for the in vitro determination of antibodies against BP180 in human serum or plasma for the diagnosis of the following diseases: bullous pemphigoid, pemphigoid gestationis, mucous membrane pemphigoid and lichen ruber pemphigoides.

Clinical significance: Bullous autoimmune dermatoses belong to organ-specific autoimmune diseases. They are characterised by the formation of autoantibodies against structure proteins of the skin. These structural proteins establish the cell-to-cell contact in ceratinocytes within the epidermis and the adhesion of the epidermis to the dermis. Bullous autoimmune dermatoses are divided in 4 main groups by means of their target antigens and the localisation of the blis-ters: pemphigoid and pemphigus diseases, epidermolysis bullosa acquisita and Duhring’s der-matitis herpetiformis. In pemphigus diseases the blisters are formed intraepidermally, whereas they occur in all other bullous autoimmune dermatoses subepidermally.

With 0.7 to 1.8 new cases per year per 100,000 inhabitants, the bullous pemphigoid (BP) is the most frequent subepidermal blister-forming autoimmune dermatosis. The disease mainly affects elderly people. The manifestation of the BP are bulging blisters at the integument. However, the BP may proceed without blisters for weeks or months. Therefore, elderly pa-tients with irritating skin disorders persisting for long periods should be tested for BP in dif-ferential diagnosis. Various immunological methods are used for differential diagnosis of the disease. The presence of circulating autoantibodies is significant in 90% of BP patients. These autoantibodies are mainly directed against 2 hemidesmosomal proteins. The BP antigens have molar masses of 180 kDa (BP180) and 230 kDa (BP230), respectively. BP230 is localised intracellularly in the hemidesmosomal plaque. BP180 is a transmembrane glycoprotein with an intracellularly local-ised C-terminus and an extracellular N-terminus. The ec-todomain consists of 15 collagenous and 16 non-collagen-ous domains. The 16th non-collagenous domain (NC16A) directly flanking the ceratinocyte membrane presents the immunogenic epitope. The majority of BP patients have autoantibodies against BP180. Direct and indirect immun-ofluorescence is used for the determination of autoanti-bodies. Tissue-bound autoantibodies and/or complement deposits in biopsies of perilesional skin can be determined using direct immunofluorescence. Circulating autoantibod-ies in the patient serum can be found by means of indirect immunofluorescence (substrate: oesophagus, primate and human skin). Autoantibodies against basement membrane show a fine linear staining between the stratum basale and the connective tissue. Autoantibody specificity can be char-acterised using monospecific ELISA or immunoblots.

Application of the Anti-BP180-NC16A-4X ELISA: The detection of autoantibodies in the skin and/or in the serum of patients is decisive in BP diagnosis. BP patients mainly have autoanti-bodies against BP180. These autoantibodies can be found in the skin using direct immunofluo-rescence. Autoantibodies circulating in the serum can be detected by means of indirect immun-ofluorescence using organ tissues. The Anti-BP180-NC16A-4X ELISA1 uses a tetramer of the immunogenic NC16A domain and is a reliable alternative for the indirect immunofluorescence test. The advantage of the ELISA is the clear characterisation of the autoantibody specificity when using the recombinant BP180 and the resulting differentiation of other bullous autoim-mune dermatoses such as pemphigus diseases, epidermolysis bullosa acquisita and Duhring’s dermatitis herpetiformis. The multimer form of the autoantigen increases the immunoreactiv-ity, thus improving the efficiency of the autoantibody test. The serum level of autoantibodies against BP180 correlates with the BP activity.

1 German Patent Application No. 10 2006 059 574.2

Panel nAnti-BP180

positive

Bullouspemphigoid (BP)

118 106 (89.8%)

Pemphigoidgestationis (PG)

20 20 (100.0%)

Asymptomatic blood donors

494 10 (2.0%)

Rheumatoid arthritis (RA)

107 2 (1.9%)

Progressive systemic sclerosis (PSS)

50 2 (4.0%)

Systemic Lupus erythematosus (SLE)

72 1 (1.4%)

Sensitivity 118 89.8%

Specificity 723 97.9%


EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Phone +49 451 58550 · Fax 5855591 · E-mail [email protected] · www.euroimmun.de










EUROLINE-WB:


Allergology:









thyroglobulin (TG)




Made in Germany

Linearity: The linearity of the ELISA was determined by assaying serial dilutions of 6 serum samples. The linear regression was calculated, R2 amounting to > 0.95 in all samples. The Anti-BP180-NC16A-4X ELISA (IgG) is linear at least in the range of 10 to 199 RU/ml.

Reproducibility: The reproducibility of the test was investi-gated by determining the intra- and inter-assay coefficients of variation using 4 sera. The intra-assay CVs are based on 20 determinations and the inter-assay CVs on 4 determina-tions performed in 6 different test runs.

Clinical sensitivity and specificity: Sera from 118 BP patients, a control panel of 229 patients with other autoimmune diseases and 494 healthy blood donors were investigated using the EUROIMMUN Anti-BP180-NC16A-4X ELISA. The sensitivity of the ELISA for BP was 90%, with a specificity of 98%. Additionally, 101 patients (> 70 years of age) with non-inflammatory skin diseases were tested, resulting in a specificity of the ELISA of 99 %.

Reference range: Levels of anti-BP180 antibodies were determined in 494 sera from healthy blood donors of between 18 and 68 years of age (185 women, 309 men) using the EUROIMMUN ELISA. The mean concentration of antibodies against BP180 was 4.5 RU/ml and the results ranged from 0.01 to 168.0 RU/ml. With a cut-off of 20 RU/ml, 2.0% of blood donors were anti-BP180 positive.

ROC analysis: In an analysis of 118 BP samples and 723 control sera the following results were achieved:

Correlation of the EUROIMMUN and MBL Anti-BP180 ELISAs: The antibody concentration was determined in 118 sera from BP patients and a control panel of 229 sera from patients with other autoimmune diseases (RA, PSS, SLE) using the EUROIMMUN and MBL Anti-BP180-ELISA. The qualitative re-sults of the ELISAs correlated in 99% (BP panel) and 98% (control panel).

Technical data:

Antigen Tetramer of the immunogenic NC16A domain (BP180), based on hu-man cDNA, expressed in E.coli



Reagents Ready for use. Exception: wash buffer (10x). Colour-coded solutions, largely exchangeable with those of other EUROIMMUN-ELISA.



Kit format 48 single break-off wells, incl. all necessary reagents.

Order no. EA 1502-4801-2 G

Test Characteristics Anti-BP180-NC16A-4X ELISA (IgG)

Intra-assay variation, n = 20

Inter-assay variation, n = 4 x 6

SerumMean value

(RU/ml)CV (%)

Mean value (RU/ml)

CV (%)

1 27 3.0 26 4.8

2 61 1.2 61 3.1

3 119 1.6 119 4.2

4 177 2.1 174 2.6

RU/ml0,1 1 10 100 1000

Freq

uen

cy n

0

20

40

60

80

100494 Blood donors

104 BP-Patienten65 BP-Patienten 104 BP-Patienten

RU/ml1 10 100 1000 10000

Freq

uen

cy n

0

2

4

6

8

10

12

14

16118 BP patients Blood donors, n = 494


Cut off 12.5 RU/ml 27.4 RU/ml


13.4 RU/ml 95 % 91 %

19.8 RU/ml 98 % 90 %

MBL1: 9.0 RU/ml 95 %2 89 %2

1 according to the guidelines of the manufacturer 2 determined in 118 BP sera and 229 control sera

n = 118(BP)

MBL Anti-BP180 ELISA (IgG)

positive negative

EUROIMMUN Anti-BP180-NC16A-4X ELISA (IgG)

pos. 105 1

neg. 0 12

n = 229(RA, PSS, SLE)

MBL Anti-BP180 ELISA (IgG)

positive negative

EUROIMMUN Anti-BP180-NC16A-4X ELISA (IgG)

pos. 5 0

neg. 7 217

ag serological diagnosis of autoimmune … against...euroimmun medizinische labordiagnostika ag...

Documents