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Autoantikörper ohne Organspezifi tät

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Organspezifi sche AutoantikörperAUTOIMMUN-

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System. Lupus erythematodes

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Habituelle Aborte

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Hashimoto-Thyreoiditis

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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 EUROIMMUN

M 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 EUROIMMUN

M 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 EUROIMMUN

M 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

Introduction

Determination of anti-mitochondrial anti-bodies (AMA) is of particular signifi cance for the diagnosis of primary biliary liver cir-rhosis (PBC), an immune-mediated chronic infl ammatory cholestatic liver disease of unknown aetiology. The most specifi c and sensitive diagnostic markers are antibod-ies against the M2 antigen. The molecular targets of these autoantibodies have been identifi ed as members of the 2-oxoacid de-hydrogenase complex family of enzymes within the mitochondrial respiratory chain. Among these enzyme components, the lipoyl binding domains (E2) are the major autoantigens in PBC.

Methods

This study reports the development of a new ELISA using an antigenic preparation based on a mixture of native M2 with a re-combinant fusion protein consisting of the lipoyl domains of BCOADC-E2, PDC-E2 and OGDC-E2 (BPO, synonym: M2-3E). The new assay called Anti-M2-3E ELISA was com-pared with a commercially available con-ventional ELISA using native PDC alone, or an ELISA based on BPO alone or an indi-rect immunofl uorescence test (IIFT) using a BIOCHIP Mosaic™ of rat kidney/stomach/liver tissue and HEp-2010 cells as substrate. Tests were performed on serum samples obtained from patients with PBC (n = 170),

autoimmune hepatitis (AIH, n = 46), PBC/AIH type 1 overlap syndrome (n = 3), chronic vi-ral hepatitis (n = 200), and from 400 healthy blood donors (HBD).

Results

At a comparable specifi city of approximate-ly 100%, use of the recombinant fusion protein BPO increased the sensitivity of the ELISA to 90.2%. By applying a mixture of native M2 and the recombinant antigen as target structures, the sensitivity of the ELISA system could be further increased to 93.1%. 99% of the samples that tested

positive in IIFT were also identifi ed by the novel Anti-M2-3E ELISA, whereas 5% of all AMA positive samples where exclusively detected by ELISA.

Discussion

The special confi guration of the BPO (M2-3E) antigen ensures simultaneous presen-tation of all three major antigens of the mitochondrial respiratory chain. Combined use of the recombinant polypeptide BPO with native M2 as substrates in one ELISA resulted in an increase in sensitivity of 14% compared to the classic anti-M2 ELISA, while preserving a specifi city of 99.6%. The new Anti-M2-3E ELISA may be used for the detection of AMA as an alternative to IIF. Moreover, it offers the potential to identify AMA in patients with suspected PBC but negative results in IIF.

Detection of primary biliary liver cirrhosis-associated anti-mitochondrial antibodies using an improved test system:

Anti-M2-3E ELISA

C. Daehnrich1, M. Mytilinaiou2, A. Rosemann1, C. Probst1, W. Schlumberger1,

D. P. Bogdanos2, W. Stoecker1, and L. Komorowski1

1Institute for Experimental Immunology, affi liated to EUROIMMUN, Luebeck, Germany2Institute of Liver Studies, King‘s College London School of Medicine, London, United Kingdom

Schematic illustration of BPO (synonym: M2-3E): The recombinant polypeptide His-BPO consists of the immunogenic lipoyl binding domains of the E2 subunit of branched-chain 2-oxoacid dehydrogenase (BCOADH), the E2 subunit of pyruvate dehydrogenase (PDH), the E2 subunit of 2-oxoglutarate dehydrogenase (OGDH) and a N-terminal His-Tag. TP: transit peptide, LB: lipoyl binding, PBC spec.: primary biliary liver cirrhosis specifi c (autoantibody binding), aa: amino acids, 3E: 3 enzymes.

BCOADH-E2

Lipoamide acyltransferase component of branched-chain alpha-keto acid dehydrogenase complex

PDH-E2

Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex

OGDH-E2

Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex

PBC spec.

118 aa

LB LBTPTP LB TP LB

BPO (M2-3E)

138 aa 82 aa

(His)8

PBC spec. PBC spec.

Test system Sensitivity Specifi city

Anti-M2-3E ELISA 93.1% 99.6%

Anti-BPO ELISA 90.2% 98.8%

Anti-M2 ELISA 79.8% 100.0%

IIFT AMA kidney (rat) 89.0% 98.8%

Anti-M2-3E ELISA

Anti-M2 ELISA IIFT (rat kidney)

all negative

11

PBC sera(n = 170)

0 10

194

4

131

Anti-M2-3E ELISA

PBC/AIHoverlap syndrome

OD

3.0

2.5

2.0

1.5

1.0

0.5

Cut-off

0.0PBC

n = 170

AIH

n = 46

HBD

n = 400

Viralhepatitis

n = 200

Introduction

Anti-neutrophil cytoplasma antibodies (ANCA) with either cANCA or pANCA fl uorescent patterns are detectable in ANCA-associ-ated vasculitis (AAV). The cANCA pattern is mainly produced by antibodies against proteinase-3 (PR3), but some cANCA posi-tive sera do not react with PR3 in different conventional ELISA systems which use inap-propriate antigen substrates and thus lack sensitivity. A new PR3 substrate was created consisting of a recombinant human PR3 de-signer antigen mixed with a native antigen, isolated from human neutrophils.

Methods

An enzymatically inactive PR3 variant was constructed 1 and, for the fi rst time, ex-pressed in human cells. By susbstitution of alanine to serine at position 176, the enzy-matic activity could be totally blocked, which made an effi cient, large-scale production of the authentic PR3 antigen in a human (!) cell line possible. A mixture of this recombinant (hr) PR3 with human native (hn) PR3 was used as coating antigen.

The resulting ELISA (Anti-PR3-hn-hr) was compared with ELISA systems, which apply directly coated substrates, either contain-ing hn-PR3 or hr-PR3, as well as a capture ELISA for PR3-ANCA. Assay characteristics were determined in AAV patients (n = 248), with special attention to those patients with

cANCA (n = 132), as well as a large cohort of disease controls (n = 585) and healthy blood donors (n = 429). Additionally, for prediction of relapses, serial samples of 46 PR3-AAV patients were analyzed.

Results

At a predefi ned specifi city of 99%, the sen-sitivity for AAV was increased to 94.7% in both ELISA systems containing the hr-PR3 antigen. For prediction of relapses by rises in antibody titers against PR3, the capture ELISA showed even slightly better results (odds ratio 12.5), closely followed by the novel Anti-PR3-hn-hr ELISA (odds ratio 8.9).

Discussion

Proteolytic damage of the transfected cell line as well as of the molecule itself is a major problem in providing the target an-tigen PR3 for immunoassays. In order to stabilize the protein, we created the hr-PR3, which lacks enzymatic activity but exhibits

all necessary conformational properties for antibody detection as well as a His-tag for easy and reliable microplate coating. As a consequence of the modern protein design, the test performance of the novel Anti-PR3-hn-hr ELISA became excellent, the sensitiv-ity even comparable to IIF, by far superior to conventional ELISA systems. The new test system is not compromised by some dis-advantages of the capture ELISA, i.e. poten-tial masking of PR3 epitopes by the murine monoclonal antibody or reporting false posi-tive reactions due to the presence of human anti-mouse antibodies. Together with the good predictability of clinical relapses, the novel Anti-PR3-hn-hr ELISA therefore serves as an ideal tool for the identifi cation and fol-low-up of PR3-positive AAV patients.

The novel Anti-PR3-hn-hr ELISA using a mixture of human native and in human cells expressed authentic recombinant PR3

J. Damoiseaux1, C. Daehnrich2, A. Rosemann2, C. Probst2, L. Komorowski2,

E. Csernok3, C. A. Stegemann4, K. Egerer5, F. Hiepe5, P. van Paassen1,

W. Stoecker2, W. Schlumberger2, and J. W. Cohen Tervaert1

1Department of Clinical and Experimental Immunology, University Hospital Maastricht, the Netherlands2Institute for Experimental Immunology, affi liated to EUROIMMUN AG, Luebeck, Germany

3Department of Rheumatology, University Hospital of Schleswig-Holstein, Luebeck, Germany4Department of Nephrology, University Medical Center Groningen, the Netherlands

5Department of Rheumatology and Clinical Immunology, Charité – Universitätsmedizin Berlin, Germany

ELISASensitivity at a

specifi city of 99%AUC

Anti-PR3-hn-hr 94.7% 0.984

Anti-PR3-hr 94.7% 0.982

Anti-PR3-hn 80.3% 0.975

Anti-PR3-Capture* 76.5% 0.961

alanin

e glu

tam

ate

dipep

tide

positio

n 176 ala

nine

(inac

tive c

entre

)

His-ta

g

maturation

mature recombinant enzymatically inactive proteinase 3, AE-PRTN3(S176A)-His

Modified maturation in non-hematopoetic cells

signal

sequen

ce

alanin

e glu

tam

ate

activ

atio

n dip

eptid

e

positio

n 176 s

erin

e

(acti

ve ce

ntre)

C-term

inal

propet

ide

maturation

mature proteinase 3

Maturation in hematopoetic cells

Test characteristics

Anti-PR3-hn-hr

Anti-PR3-hr

Anti-PR3-hn

Anti-PR3-

Capture

Rises in relapses (n = 23) 19 14 16 20

Rises in stable patients (n = 23)

8 5 8 8

Sensitivity (%)with respect to relapses

83 61 70 87

Specifi city (%)with respect to relapses

65 78 65 65

PPV (%) 70 74 67 71

NPV (%) 79 67 68 83

Odds ratio 8.9 5.6 4.3 12.5

P-value 0.002 0.016 0.038 0.001

*Sensitivity 85.6% at a specifi city of 98%

1Sun J, Specks U et al. (1998) Clin Exp Immunol 114:320-6

Scientifi c presentation at the 10th International Workshop on Autoantibodies and Autoimmunity (Guadalajara, Mexico, March 2008)

Introduction

Coeliac disease (CD) is characterized by IgA and IgG antibodies against native gliadin (anti-n-gliadin), antibodies against tissue transglutaminase (anti-tTG) and endomysi-um antibodies (EmA). Generally, anti-n-glia-din are less accurate than anti-tTG. Recently, specifi c antibodies against synthetic peptides derived from partially deamidated gamma-gliadin were described1. We present a new ELISA, in which the antigen represents a re-petitive motive of the described peptides.

Methods

The synthetic DNA coding for GAF(3X), which includes threefold repetitive modi-fi ed copies of PLQPEQPFP and PEQLPQFEE, was cloned into a prokaryotic expression vector and expressed in E. coli. Microplates were coated with the GAF(3X) preparation. After incubation with patients sera, bound antibodies were detected by peroxidase-labelled anti-human IgA and IgG. For com-parison, anti-n-gliadin, anti-tTG and EmA

(all reagents obtained from EUROIMMUN, Germany) were measured. Sera of 181 chil-dren with biopsy-proven CD (71 males, 110 females, age range 1-17 years, mean age 7.24 years) under normal gluten-containing diet (blood withdrawal at time of biopsy) and of 220 biopsied disease controls (120 males, 100 females, mean age 9.38 years) were ana-lyzed. The diagnostic validity of the test was assessed by receiver-operating characteris-tic (ROC) analysis and area under the ROC curve (AUC) was calculated (in %). Diagnos-tic accuracy was calculated as the sum of true positives and true negatives divided by the total number of tested individuals.

Results

AUC was signifi cantly larger (p < 0.001) for anti-GAF(3X) (IgG 98.3%; IgA 94.8%) than for anti-n-gliadin (IgA 88.1%; IgG 86.5%). There was no signifi cant difference (p > 0.42) in AUC between IgG anti-GAF(3X), IgA EmA (97.5%) and IgA anti-tTG (97.9%). Diagnostic accuracy was 94.8% for IgG-GAF(3X), 94.0% for IgA EmA and 96.0% for IgA tTG.

Discussion

The new anti-GAF(3X) ELISA is based on a completely new antigen2, which refl ects a peptide derived from native gliadin by deam-idation through tTG. Therefore, it re presentsa very specifi c epitope for antibodies in CD. Very interestingly, IgG anti-GAF(3X) are more accurate than IgA anti-GAF(3X), and there was no signifi cant difference in the AUC compared to IgA anti-tTG or IgA EmA. The novel Anti-GAF(3X) ELISA refl ects a new gold standard in the serological diagnosis of CD and offers the potential to identify also IgA defi cient CD patients, since specifi c IgG antibodies are preferably tested.

The new Anti-GAF(3X) ELISA: Highly reliable serologic diagnosis of childhood coeliac disease

C. Prause1, C. Probst2, L. Komorowski2, C. Daehnrich2, W. Schlumberger2,

T. Richter3, A. C. Hauer4, M. Stern5, H. Uhlig6, M. Laass7,

K.-P. Zimmer8, and T. Mothes1

1Institute for Laboratory Medicine at the University clinic of Leipzig, Germany2Institute for Experimental Immunology, affi liated to EUROIMMUN, Luebeck, Germany

Children‘s clinics at the Municipal clinic of Leipzig3 and the University clinics of Graz4, Austria, Tuebingen5,Leipzig6, Dresden7 and Giessen-Marburg8, Germany

Peptide 1 Peptide 2

GAF-1 GAF-2 GAF-3

Sequence optimizationand assembly

Chemical synthesis

Expressionin E. coli

DNA sequence coding for gliadin analogous peptides

GAF (gliadin analogous fusion peptide)

GAF(3X)

Schematic illustration of GAF(3X) ROC curves of 7 different antibody tests. For sake of clarity, only 60 to 100% sensitivity and 40 to 100% specifi city are shown.

Se

nsit

ivit

y (

%)

IgA Anti-GAF(3X)

IgA Anti-n-gliadin

IgA Anti-tTG

IgA EmA

IgG Anti-GAF(3X)

IgG Anti-n-gliadin

IgG Anti-tTG

100 - Specificity (%)

0 20 40 0 20 40 60

60

80

100

Literature: 1Schwertz et al. Serological assay based on gliadin-related nonapeptides as a highly sensitive and specifi c diagnostic aid in celiac disease. Clin Chem 2004; 50: 2370-2375. 2Probst C, Daehnrich C, Schlumberger W, Stoecker W, Komorowski L, Mothes T. DE 10 2007 025 291.0 (2007)

Scientifi c presentation at the 10th International Workshop on Autoantibodies and Autoimmunity (Guadalajara, Mexico, March 2008)

Anti-GAF(3X) Anti-tTG Number of CD patients

IgA IgG IgA (total: 181)+ + + 157+ + - 2- + + 11- + - 2- - + 5- - - 4

Anti-GAF(3X) Anti-tTG Number of controls

IgA IgG IgA (total: 220)+ + + 4+ + - 3+ - + 1+ - - 16- + - 5- - + 2- - - 189

Anti-n-gliadin Anti-GAF(3X) Anti-tTG EmA

IgA IgG IgA IgG IgA IgG IgA

AUC (%) 88.1 86.5 94.8 98.3 97.9 93.0 97.5

Sensitivity (%) at 95% specifi city 54.1 30.9 82.9 93.9 95.6 61.9 92.2

Based on cut-off calculated for maximum sum of sensitivity plus specifi city

Sensitivity 78.5 81.2 87.9 95.0 95.6 82.3 96.7

Specifi city 80.0 79.1 89.1 94.6 96.4 86.8 91.8

Number of false negatives 44 46 24 12 8 29 4

Number of false positives 39 34 22 9 8 32 17

Diagnostic accuracy (%) 79.3 80.1 88.5 94.8 96.0 84.8 94.0

Introduction

Autoantibodies against exocrine pancreas (PAb) have been reported to be pathog-nomonic markers of Crohn‘s disease (CD), with a prevalence of 39% in the indirect im-munofl uorescence test (IIFT). Two different patterns were observed: a reticulogranular and a droplet pattern in the pancreatic acini (Stoecker W et al., Scand J Gastroenterol, 1987). Based on earlier fi ndings, we hy-pothesized that the corresponding antigens would be glycosylated and used a new strategy to identify them.

Antigen Identifi cation Strategy

Human pancreas was homogenized at alka-line pH and ultracentrifuged. The cell-free pancreas supernatant generated thereby was still able to neutralize PAb in IIFT when tested in the presence of high concentra-tions of protease inhibitors, which were ap-plied to prevent tissue digestion. In the next step, the supernatant was immobilized on microtiter plates pre-coated with lectins of different known specifi cities, which allowed

selected capturing of glycosylated antigens. Subsequently, an ELISA protocol was con-ducted using PAb positive and negative control sera and anti-human-IgG horserad-ish peroxidase conjugate. By employing this strategy, the most striking specifi c re-activity of PAb positive sera was observed in wells where the cell-free supernatant was immobilized to the plate via Ulex euro-paeus agglutinin I (UEA I). This lectin was then used to purify the corresponding glyco-proteins from pancreas supernatant by af-fi nity chromatography. The eluate thereof revealed two protein bands in SDS-PAGE and was able to neutralize PAb reactivity in IIFT. Subsequently, antigen-containing eluate fractions were prepared by SDS-PAGE and tryptic cleavage for MALDI-ToF fi ngerprinting. Here, the two proteins were identifi ed as the proteoglycans CUB/zona pellucida-like domain-containing protein CUZD1 (Acc. No.: Q86UP6, UniProt) and pancreatic zymogen granule membrane major glycoprotein GP2 (Acc. No.: P55259, UniProt). cDNA for both antigens was in-tegrated into eukaryotic expression vectors and transfected into a human embryonic kidney cell line (HEK293). Finally, the cells

were applied in parallel to frozen pancreas sections as substrates for indirect immuno-fl uorescence. Reactivities were tested by applying 20 characterized PAb positive sera from patients with CD and 100 PAb negative controls (30 patients with ulcerative colitis and 70 healthy blood donors). In all of the CD sera, reactions of the CUZD1 transfected cells were concordant with the pancreatic reticulogranular pattern, reactions of GP2 cells with the droplet pattern (Figures).

Discussion

We used a screening ELISA to analyze the abilities of a set of lectins to capture the PAb corresponding antigens from cell-free pancreas supernatant. The fucose-specifi c UEA I turned out to be the best-suited lectin to purify the antigens, which allowed their identifi cation by MALDI-ToF. The concord-ant reaction of the native and recombinant substrates guarantees the unequivocal au-thenticity of the two identifi ed CD-related autoantigens. We conclude that the long sought pancreatic autoantigens in Crohn‘s disease are now identifi ed.

Identifi cation of two different proteoglycans from exocrine pancreas as the long sought-after autoantigens

in Crohn‘s disease: CUZD1 and GP2

W. Stoecker1, M. O. Glocker2, C. Probst1, B. Teegen1, A. Friedrich1,

S. Sokolowski1, and L. Komorowski1

1Institute for Experimental Immunology, affi liated to EUROIMMUN AG, Luebeck, Germany2University of Rostock, Proteome Center, Germany

Pancreas, reticulogranular pattern HEK293-CUZD1 Pancreas, droplet pattern HEK293-GP-2

Scientifi c presentation at the 6th International Congress on Autoimmunity (Porto, Portugal, September 2008)

Introduction

Until now, antibodies against dsDNA have been the most important markers for the diagnosis of systemic lupus erythemato-sus (SLE) and for the determination of dis-ease activity, especially in lupus nephritis. In many conventional ELISA systems, the target antigen dsDNA is linked to the solid phase by means of poly-L-lysine or pro-tamine sulphate. However, these sub stancesare prone to cause unspecifi c reactions.

As one result of our long-standing re-search on nucleosomes, we experienced their pronounced adhesiveness to plastic surfaces as well as their high affi nity to pure dsDNA. The novel Anti-dsDNA-NcX ELISA takes advantage of the capability of nucleosomes to link DNA to microtiter plates.

Methods

Purifi ed nucleosomes, free of Scl70, histone H1 and other non-histone components 1,were used as an ultra-thin adhesive layer for the immobilisation of dsDNA in the Anti-dsDNA-NcX ELISA (IgG). This test sys-tem was compared to a conventional Anti-dsDNA ELISA, the Farr-RIA (gold standard, IgA/G/M), an Anti-Nucleosomes ELISA (IgG) and indirect immunofl uorescence using

Crithidia luciliae (IFA, IgG), all reagents from EUROIMMUN, Germany. The study comprised 208 patients with SLE, 88 with Sjögren‘s syndrome (SS) and 81 with pro-gressive systemic sclerosis (PSS).

Results

At a predefi ned specifi city of 98%, the sen-sitivity of the novel Anti-dsDNA-NcX ELISA was increased to 60.8%, clearly exceeding the conventional Anti-dsDNA ELISA and the Anti-dsDNA RIA (data based on ROC analy-sis). With regard to SS and PSS, the new test system reached a maximum specifi city of 98.2% for SLE. For comparison, the sen-sitivity of IFA was 27.4% at a specifi city of 96.4%. However, all six control sera, which reacted exclusively in IFA, originated from patients with PSS, which frequently show

an overlap with SLE: possibly, IFA is more suitable to identify such cases than the oth-er two test systems.

Discussion

The special confi guration of the Anti-dsDNA-NcX ELISA ensures clear presentation of the major dsDNA epitopes and minimizes false positive reactions. Of all the individual assays tested, the Anti-dsDNA-NcX ELISA revealed the highest sensitivity in the diagnosis of SLE. RIA and IFA might be used to identify some remaining SLE patients with negative ELISA results (7.2% in our study). In sum, our data demonstrate that the Anti-dsDNA-NcX ELISA is superior to the Farr-RIA and to the IFA us-ing Crithidia luciliae, and has the potential to replace them as gold standards in the sero-logical diagnosis of SLE.

Anti-dsDNA-NcX ELISA: Superior to Farr-RIA and IFA using Crithidia luciliae for SLE diagnosis

Robert Biesen1, Cornelia Daehnrich2, Anke Rosemann2, Falk Hiepe1, Karl Egerer1,

Winfried Stoecker2, Wolfgang Schlumberger2

1Department of Rheumatology, Charité - Universitätsmedizin Berlin, Germany2Institute for Experimental Immunology, affi liated to EUROIMMUN AG, Luebeck, Germany

Anti-dsDNA-NcXELISA

Anti-dsDNAELISA

Anti-dsDNARIA

Anti-NucleosomesELISA

AUC 0.85 0.82 0.85 0.83

CI 0.81 - 0.89 0.77 - 0.86 0.81 - 0.89 0.79 - 0.87

Sensitivity at a specifi city of 95%

65.1% 49.3% 54.1% 54.1%

Sensitivity at a specifi city of 98%

60.8% 35.4% 53.1% 52.6%

Sensitivity at a specifi city of 99%

57.4% 34.9% 52.2% 52.6%

Maximal sum of sensitivity and specifi city

161.3% 152.6% 156.0% 151.8%

Anti-dsDNA-NcX ELISA

IU/m

l

0

200

400

600

800

1000

Cut-off

SLE SS PSS HC

n = 208 n = 88 n = 81 n = 400

Literature: 1Suer W, Daehnrich C, Schlumberger W, Stoecker W. Autoantibodies in SLE but not in scleroderma react with protein-stripped nucleosomes. J Autoimmun 22 (2004) 325-334

27

9

49

46

33

Anti-dsDNA-NcX ELISA

Anti-dsDNA RIA(Farr)

IFA (Crithidia luciliae)

2all negative

69

SLE sera(n = 208) Anti-dsDNA-NcX ELISA

Anti-dsDNA RIA(Farr)

IFA (Crithidia luciliae)

all negative

158

Control sera(n = 169)

2** 6*0

00

3***

0

* 6 PSS ** 2 PSS *** 2 PSS, 1 SS

Scientifi c presentation at the 10th International Workshop on Autoantibodies and Autoimmunity (Guadalajara, Mexico, March 2008)

Introduction

The indirect immunofl uorescence test (IIFT) is considered the gold standard for the detection of antibodies against neutrophil granulocytes (ANCA). The EUROPLUSTM system enables to combine cells or tissues with substrates of single antigen microdrops, e.g. granulocytes with MPO and PR3 dots in one incubation fi eld. In the present study we evaluated the diagnostic applicability of this new system to ANCA-associated vasculitis (AAV).

Methods

An AAV cohort consisting of 248 cases (114 biopsy-proven AAV patients, 74 AAV patients in our outpatient clinic, and 60 Wegener’s granulomatosis patients) as well as 112 con-trol samples (other forms of vasculitis n = 55, rheumatoid arthritis n = 30, healthy controls n = 27) were analysed by IIFT using the EUROPLUSTM BIOCHIP Mosaic 23 (see Fig. 2). Samples were only classifi ed as pANCA positive if a positive result was obtained for both ethanol and formalin fi xed granulocytes. The reference tests were Anti-PR3-hn-hr and Anti-MPO ELISA (EUROIMMUN AG).

Results

In a mixed AAV cohort, the observed cAN-CA and pANCA patterns were supported by a positive reaction of the PR3 or MPO dots, respectively, in 128 (97%) and 63 (93%) of the cases. Of 116 cANCA negative and 180 pANCA negative cases, 115 (99%) and 178 (99%) were confi rmed by the dots not to contain anti-PR3 or anti-MPO autoanti-bodies. The negative dot results of the 4 cANCA positive cases were confi rmed by ELISA. One case was PR3 dot positive and cANCA negative: the positive dot result

was in agreement with ELISA. Four of the 5 MPO dot negative cases that were pANCA positive were also negative with the Anti-MPO ELISA. In one of the 2 pANCA nega-tive but MPO dot positive cases the pres-ence of anti-MPO antibodies was confi rmed by ELISA. For all 109 control samples that showed neither a cANCA nor a pANCA pat-tern the absence of PR3-ANCA and MPO-ANCA was confi rmed by the antigen dots. The antigen dots also confi rmed the ab-sence of anti-PR3 and anti-MPO antibodies in agreement with the ELISA results in all diffi cult cases of both cohorts that showed an atypical ANCA pattern (about 12%).

Discussion

EUROPLUSTM BIOCHIPS signifi cantly facili-tate the interpretation of IIFT results. In the vast majority of cases the easily interpret-able results of the PR3 and MPO antigen dots support the ANCA pattern observed on the conventional cell substrates. In the dis-crepant cases, the dots provide additional diagnostic information: Negative results for the antigen dots help to identify cases where cANCA and pANCA are directed against anti-gens other than PR3 and MPO. Alternatively, the dots help to identify anti-PR3 and anti-MPO antibody positive samples that would have been missed by interpretation of the ANCA pattern only, e.g. because both, anti-PR3 and anti-MPO antibodies are present in the sample. The fi nal diagnosis of specifi c autoantibodies can be obtained after just one incubation since the EUROPLUSTM dots already lead to antigen specifi c results that correlate very well with other monospecifi c tests. However, for quantitative results, a corresponding ELISA has to be performed.

EUROPLUSTM ANCA BIOCHIP Mosaic: MPO and PR3 antigen dots improve the detection of ANCA

by indirect immunofl uorescenceJ. Damoiseaux1, M. Buschtez2, U. Steller2, B. Zerbe2, A. Rosemann2,K. Fechner2, W. Schlumberger2, J.W. Cohen Tervaert1, W. Stoecker2

1Department of Clinical and Experimental Immunology, University Hospital Maastricht, the Netherlands2Institute of Experimental Immunology, affi liated to EUROIMMUN AG, Luebeck, Germany

AAV cohort, n = 248IIFT, cANCA

positive negative

EUROPLUS

PR3 dot

positive 128 1

negative 4 115

Antigen solution

Cover glasses

BIOCHIPs EUROPLUSTM

slide

Tissue

Antigen dots

Cells

Fig. 2b: EUROPLUSTM Granulocyte Mosaic (granulocytes EOH, primate liver, granulocytes HCHO, HEp-2, PR3, MPO): pANCA, MPO-positive.

Fig. 2a: EUROPLUSTM Granulocyte Mosaic (granulocytes EOH, primate liver, granulocytes HCHO, HEp-2, PR3, MPO): cANCA, PR3-positive.

Fig. 1

AAV cohort, n = 248IIFT, pANCA

positive negative

EUROPLUS

MPO dot

positive 63 2

negative 5 178

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

*PNS-definierende Antikörper

Aus: Wandinger KP. Paraneoplastische Neurologische Syndrome. Klinik und Labordiagnostik. MTA-Dialog 2008: 266-270

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.

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

(RU

/ml)

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

eas e

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

Antikörper

(Synonym)

Antigen

(Molekularmasse)Fluoreszenzmuster

Paraneoplastisches

Syndrom

Häufigste

Tumore

Anti-Hu(ANNA-1)*

Hu-Protein(38 kDa)

Zellkerne zentraler und peripherer Neurone

EnzephalomyelitisSensible Neuropath.

SCLC, Neuroblastom

Anti-Yo (PCA-1)*

CDR2, CDR62 (34 kDa und 62 kDa)

Cytoplasma der Purkinje-zellen

Kleinhirndegeneration Ovar, Mamma, Uterus

Anti-Ri (ANNA-2)*

NOVA (55 kDa und 80 kDa)

Zellkerne zentraler Neurone

Opsoklonus-Myoklo-nus-Syndrom

Mamma, SCLC

Anti-PNMA1 (Ma1)*

Ma-Protein (37 kDa)

Nervenzellnukleoli Rhombenzephalitis (Hirnstamm)Limbische Enzephal.

Mamma, ver-schiedene

Anti-PNMA2 (Ma2/Ta)*

Ma-Protein(40 kDa)

Nervenzellnukleoli Rhombenzephalitis (Hirnstamm)Limbische Enzephal.

Hoden

Anti-Amphi-physin*

Amphiphysin(128 kDa)

Präsynaptische Nerven-enden des Kleinhirns

Stiff-Person-Syndrom Mamma, SCLC

Anti-CV2 (Anti-CRMP5)*

CRMP5 (66 kDa)

Cytoplasma der Oligo-dendrocyten

Enzephalitis SCLC, Thymom

PCA-2 Purkinjezellprotein (280 kDa)

Cytoplasma und Dendri-ten der Purkinjezellen

EnzephalitisNeuropathie

SCLC

Anti-Tr (PCA-Tr)

Unbekannt Cytoplasma der Pur-kinjezellen, punktartige Färbung der Molekular-schicht

Kleinhirndegeneration M. Hodgkin

Anti-mGluR1 Metabotroper Glutamatrezeptor (ca. 140 kDa)

Cytoplasma der Purkinje-zellen

Kleinhirndegeneration M. Hodgkin

Anti-Recoverin Recoverin (23 kDa und 65 kDa)

Photorezeptoren der Retina

Retinopathie SCLC

Anti-Titin Titin Quergestreifte Muskulatur Myasthenia gravis Thymom

Anti-GAD Glutamat-decarboxylase (65 kDa und 67 kDa)

Körnerschicht des Klein-hirns und Inselzellen (Pankreas)

Stiff-Person-Syndrom Mamma, SCLC, Kolon

Autoantikörper bei Paraneoplastischen Neurologischen Syndromen (PNS)