Differentiated ANCA diagnostics using BIOCHIP...

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EUROIMMUN Medizinische Labordiagnostika AG EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail [email protected] Differentiated ANCA diagnostics using BIOCHIP Mosaics PR3 microdroplets Granulocytes (EOH) Granulocytes (HCHO) MPO microdoplet Primate liver HEp-2 cells + Gran. (EOH) Example: Antibodies against MPO and homogeneous ANA pattern

Transcript of Differentiated ANCA diagnostics using BIOCHIP...

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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 AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail [email protected]

Differentiated ANCA diagnosticsusing BIOCHIP Mosaics

PR3 microdroplets

Granulocytes (EOH)

Granulocytes (HCHO)

MPO microdoplet

Primate liver

HEp-2 cells + Gran. (EOH)

Example: Antibodies against MPO and homogeneous ANA pattern

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EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail [email protected]

Granulo-cytes (EOH)

Granulo-cytes (HCHO)

PR3 micro-droplets

MPO micro-droplets

Primate liver

Immediatediagnosis with EUROIMMUN

BIOCHIPs: pANCA positive,

anti-MPO positive

Result: pANCA?

Anti-MPO?ANA? Granulocytes (EOH)

Final result: formalin-resistant pANCA, anti-MPO (Anti-PR3 ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-PR3 Capture ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-PR3-hn-hr ELISA: 3 RU/ml (NV < 20 RU/ml), Anti-MPO ELISA: 56 RU/ml (NV < 20 RU/ml), Anti-BPI/Elastase/Cathepsin-G ELISA: negative, EUROLINE ANA Profile 3: negative)

HEp-2 cells +gran. (EOH)

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EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail [email protected]

Granulo-cytes (EOH)

Granulo-cytes (HCHO)

PR3 micro-droplets

MPO micro-droplets

Primate liver

Immediatediagnosis with EUROIMMUN

BIOCHIPs: cANCA positive,

anti-PR3-positive

Result: cANCA?

Anti-PR3? Granulocytes (EOH)

Final result: cANCA, anti-PR3 (Anti-PR3 ELISA: > 200 RU/ml (NV < 20 RU/ml), Anti-PR3 Capture ELISA: > 200 RU/ml (NV < 20 RU/ml), Anti-PR3-hn-hr ELISA: > 200 RU/ml (NV < 20 RU/ml), Anti-MPO ELISA: 9 RU/ml (NV < 20 RU/ml), Anti-BPI ELISA: positive, Anti-Elastase/Cathepsin-G ELISA: negative, EUROLINE ANA Profile 3: negative)

HEp-2 cells +gran. (EOH)

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EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail [email protected]

Granulo-cytes (EOH)

Granulo-cytes (HCHO)

PR3 micro-droplets

MPO micro-droplets

Primate liver

Result: pANCA?

Anti-MPO?ANA?

Quickdiagnosis with EUROIMMUN

BIOCHIPs: ANCA positive, further testing

required

Granulocytes (EOH)

Final result: formalin-sensitive pANCA, anti-cathepsin G (Anti-PR3 ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-PR3 Capture ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-PR3-hn-hr ELISA: 12 RU/ml (NV < 20 RU/ml), Anti-MPO ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-BPI/Elastase ELISA: negative, Anti-Cathepsin-G ELISA: positive, EUROLINE ANA Profile 3: negative)

HEp-2 cells +gran. (EOH)

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EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail [email protected]

Granulocytes (EOH)

Granulo-cytes (EOH)

Granulo-cytes (HCHO)

PR3 micro-droplets

MPO micro-droplets

Primate liver

Immediatediagnosis with EUROIMMUN

BIOCHIPs: anti-lysoso-mes positive

Result: cANCA?

Anti-PR3?

Final result: anti-lysosomes (Anti-PR3 ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-PR3 Capture ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-PR3-hn-hr ELISA: 3 RU/ml (NV < 20 RU/ml), Anti-MPO ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-BPI/Elastase/Cathepsin-G ELISA: negative, EUROLINE ANA Profile 3: anti-dsDNA borderline)

HEp-2 cells +gran. (EOH)

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EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail [email protected]

Granulo-cytes (EOH)

Granulo-cytes (HCHO)

PR3 micro-droplets

MPO micro-droplets

Primate liver

Immediatediagnosis with EUROIMMUN

BIOCHIPs: anti-nuclear membrane

positive

Result: pANCA?

Anti-MPO?ANA? Granulocytes (EOH)

Final result: anti-nuclear membrane, anti-Ro-52 (Anti-PR3 ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-PR3 Capture ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-PR3-hn-hr ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-MPO ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-BPI/Elastase/Cathepsin-G ELISA: negative, EUROLINE ANA Profile 3: anti-Ro-52 positive)

HEp-2 cells +gran. (EOH)

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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 AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail [email protected]

Granulo-cytes (EOH)

Granulo-cytes (HCHO)

PR3 micro-droplets

MPO micro-droplets

Primate liver

Quickdiagnosis with EUROIMMUN

BIOCHIPs: granular pattern,

further testing required

Result: pANCA?

Anti-MPO?ANA? Granulocytes (EOH)

Final result: anti-RNP, anti-Ro-52 (Anti-PR3 ELISA: 4 RU/ml (NV < 20 RU/ml), Anti-PR3 Capture ELISA: 4 RU/ml (NV < 20 RU/ml), Anti-PR3-hn-hr ELISA: 9 RU/ml (NV < 20 RU/ml), Anti-MPO ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-BPI/Elastase/Cathepsin-G ELISA: negative, EUROLINE ANA Profile 3: anti-RNP positive, anti-Ro-52 positive)

HEp-2 cells +gran. (EOH)

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EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail [email protected]

Granulo-cytes (EOH)

Granulo-cytes (HCHO)

PR3 micro-droplets

MPO micro-droplets

Primate liver

Immediatediagnosis with EUROIMMUN

BIOCHIPs: pANCA pos.,

anti-MPO pos.,ANA-pos.

Result: pANCA?

Anti-MPO?ANA? Granulocytes (EOH)

Final result: formalin-resistant pANCA, anti-MPO, ANA homogeneous, anti-Ro-52 (Anti-PR3 ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-PR3 -hn-hr ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-MPO ELISA: 126 RU/ml (NV < 20 RU/ml), Anti-BPI/Elastase/Cathepsin-G ELISA: negative, EUROLINE ANA Profile 3: negative)

HEp-2 cells +gran. (EOH)

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EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail [email protected]

Granulo-cytes (EOH)

Granulo-cytes (HCHO)

PR3 micro-droplets

MPO micro-droplets

Primate liver

Result: cANCA?

Anti-PR3?

Quickdiagnosis with EUROIMMUN

BIOCHIPs: cytopl. pattern,further testing

required

Granulocytes (EOH)

Final result: anti-ribosomal P proteins (Anti-PR3 ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-PR3 Capture ELISA: 4 RU/ml (NV < 20 RU/ml), Anti-PR3-hn-hr ELISA: 5 RU/ml (NV < 20 RU/ml), Anti-MPO ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-BPI/Elastase/Cathepsin-G ELISA: negative, EUROLINE ANA Profile 3: anti-rib. P-proteins positive)

HEp-2 cells +gran. (EOH)

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EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail [email protected]

Granulo-cytes (EOH)

Granulo-cytes (HCHO)

PR3 micro-droplets

MPO micro-droplets

Primate liver

Immediatediagnosis with EUROIMMUN

BIOCHIPs: anti-centro-

meres positive

Result: pANCA? cANCA? Granulocytes (EOH)

Final result: anti-centromeres (Anti-PR3 ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-PR3 Cap-ture ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-PR3-hn-hr ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-MPO ELISA: 2 RU/ml (NV < 20 RU/ml), Anti-BPI/Elastase/Cathepsin-G ELISA: nega-tive, EUROLINE ANA Profile 3: anti-centromere protein B positive)

HEp-2 cells +gran. (EOH)

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EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail [email protected]

Granulo-cytes (EOH)

Granulo-cytes (HCHO)

PR3 micro-droplets

MPO micro-droplets

Primate liver

Result: pANCA? cANCA?

Immediatediagnosis with EUROIMMUN

BIOCHIPs: ANCA, anti-PR3 and anti-MPO

positive

Granulocytes (EOH)

Final result: ANCA, anti-PR3 and anti-MPO positive (Anti-PR3-hn-hr ELISA: 200 RU/ml (NV < 20 RU/ml), Anti-MPO ELISA: 450 RU/ml (NV < 20 RU/ml), Anti-BPI/Elastase/Cathe-psin-G ELISA: negative, EUROLINE ANA Profile 3: negative)

HEp-2 cells +gran. (EOH)

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EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail [email protected]_1200_I_UK_B04, 11/2008

Pure guesswork using ethanol-fixed granulocytes alone!

Granulocytes (EOH)

Granulocytes (HCHO)

HEp-2 cells+

granulocytes (EOH)

Primate liver

PR3 micro-droplets

MPO micro-droplets

ANCA diagnostics

Immediate reliable results using BIOCHIP Mosaics!

Granulocytes (EOH)

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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.

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

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)

FA_1201_I_UK_A01, 10/2007

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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

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

EUROIMMUN IIFTAutoimmune Diagnostics

tissue/cell substrates:adrenal gland, monkeybladder, ratcartilage (trachea), monkey*cerebellum, monkeycerebrum, monkeyCrithidia luciliae erythrocytes, human*eye, monkey*granulocytes, human (ethanol-fixed) granolocytes, human (formaldehyde-fixed)granulocytes, human (methanol-fixed)heart, monkeyHEp-2 cellsHEp-20-10 cellsHUVEChypothalamus, monkey*intestine, monkeykidney, monkeykidney, mousekidney, ratlacrimal gland, monkeylip, monkey*liver, monkeyliver, mouseliver, ratlobus temporalis, monkey*lung, monkeylymph nodes, monkey*lymphocytes, human*mamma, monkey*mouth mucosa, monkey*nerve, monkeyoesophagus, monkeyoesophagus, ratovary, monkeypancreas, monkeyparathyroid gland, monkeyparotid gland, monkeypituitary gland, monkeyplacenta, monkey*prostate, monkeySaccharomyces cerevisiaeskeletal muscle, monkeyspermatozoa, humanspleen, monkey*spinal cord, monkeystomach, monkeystomach, mousestomach, ratsynovium, monkeytestis, monkeythrombocytes, humanthymus, monkeythyroid gland, monkeytongue, monkeyVSM47 cellsumbilical cord, human

EUROPLUS™ substrates:AIH (LC-1 + SLA/LP)AMA-M2gliadinintrinsic factormyeloperoxidase (MPO)PBC (AMA M2 + Sp100)proteinase 3 (PR3)ribosomal P-proteins + Jo-1SS-A + SS-BSS-B + ribosomal P-proteins + Jo-1SS-B + Scl-70 + Jo-1thyroglobulin (TG)

BIOCHIP MosaicsTM:ANA global test: HEp-20-10/monkey liverAutoantibody Profile: combination of30 different tissues per slide CIBD Profile: monkey pancreas/intest. goblet cells (culture)/granulocytes (EOH)/Saccharomyces cerevisiaeBasic Profile: HEp-20-10/monkey liver/rat kidney/rat stomachEUROPLUS endomysium + gladin: monkey intestine/monkey liver/gliadinGranulocyte Mosaic: granulocytes (EOH)/granulocytes (HCHO)/HEp-2/monkey liverLiver Mosaic: HEp-2/monkey liver/rat liverrat kidney/rat stomach/monkey heartNeuronal Antibody Screen: monkey cerebellum/monkey nerve/monkey intestinePolyendocrinopathy Mosaic:  monkey thyroid/ monkey pancreas/monkey adrenal/monkey ovary/monkey testis/monkey stomach

Other mosaics also available Special substrate combinations on request

* Currently not available as IVD in the EU.

Made in Germany

Indications: Test system for the in vitro determination of antibodies against neutrophil granu-locytes  in human serum or plasma  for  the diagnosis of  the  following diseases:  vasculitides, microscopic polyangiitis,  rapid-progressive glomerulonephritis, Churg Strauss  syndrome, ul-cerative  colitis,  differential  diagnostics  of  Wegner’s  granulomatosis  and  systemic  lupus  ery-thematosus.

Clinical significance: ANCA are autoantibodies directed against antigens found in cytoplasmic granules of neutrophils and monocytes. Several methods are used for the detection of ANCA. The  standard  technique  is  the  indirect  immunofluorescence  (IIFT)  on  ethanol-fixed  granulo-cytes.  At  least  two  different  staining  patterns  can  be  differentiated:  a  granular  fluorescence which  is  distributed  equally  over  the  entire  cytoplasm  of  the  granulocytes,  leaving  the  cell nuclei  free  (cANCA:  cytoplasmic  pattern),  and  a  predominantly  smooth,  partly  fine  granular fluorescence wrapped ribbon-like around the cell nuclei of  the granulocytes (pANCA: perinu-clear pattern).

ANCA are typically found in Wegener’s granulomatosis (WG), microscopic polyangiitis (MPA) including renal  limited vasculitis, and Churg-Strauss syndrome (CSS), which are all  forms of small-vessel vasculitis. These three diseases are grouped together as ANCA-associated vascu-litides (AAV) according to the widely accepted classification system introduced by the Chapel Hill Consensus Conference. Classical cANCA are almost always directed against proteinase 3 (PR3) and very rarely against myeloperoxidase (MPO) or against PR3 and MPO simultaneously. They are specific for WG – a febrile, chronic granulomatous disease of the nasopharynx, lungs and  kidneys.  During  the  active  stage  the  prevalence  of  cANCA  amounts  to  more  than  90% and in the remission stage to 30 to 40%. Some cANCA exhibit a flat homogenous cytoplasmic staining in IIF (mostly termed atypical cANCA) which is often directed against bactericidal/per-meability increasing protein (BPI).

MPO is the main antigen of pANCA in patients with MPA and CSS. The autoantibodies can also be detected in patients with a wide range of non-vascular diseases, e.g.  inflammatory bowel diseases, primary sclerosing cholangitis, autoimmune liver diseases, collagenoses, rheumatoid arthritis, malignant tumours and infections. In addition to MPO, other proteins have also been identified as pANCA target antigens: lactoferrin, elastase, BPI, cathepsin G, lysozyme and beta-glucuronidase. 

All autoantibodies against neutrophil granulocytes can be investigated using indirect immun-ofluorescence. The International Consensus Statement recommends screening for ANCA using IIF and the confirmation of IIF-positive sera with both Anti-PR3 and Anti-MPO ELISA.

Application of the EUROPLUS™ “Granulocyte Mosaic 23”: The IIFT is considered as the gold standard for the determination of antibodies against neutrophil granulocytes. For the diagnosis of ANCA-associated vasculitides, a positive ANCA result  in  IIFT should be confirmed using a monospecific  Anti-PR3  or  MPO  test.  The  EUROPLUS™  technique  allows  the  combination  of conventional cell culture substrates with defined single antigens on one test field. This signifi-cantly facilitates the interpretation of pANCA, cANCA and atypical ANCA fluorescence patterns. Diagnosis  can  be  established  directly  after  the  first  incubation  because  the  screening  result (granulocytes) is confirmed straight away with the specific antigen in one and the same test. For the quantification of results, the appropriate ELISA should be performed.

EUROPLUS™ “Granulocyte Mosaic 23“

Granulocytes (EOH)

Granulocytes (HCHO)

PR3 Liver HEp-2 cells MPO

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

EUROIMMUN IIFTInfectious Serology

Viruses:AdenovirusesChikungunya virusCoxsackievirusesCrimean Congo fever virus (CCHFV)*Cytomegalovirus (CMV)Dengue viruses types 1-4 (DENV)ECHO virusEpstein-Barr virus capsid antigen (EBV-CA)Epstein-Barr virus early antigen (EBV.EA)Epstein-Barr virus nuclear antigen (EBNA)Hantaviruses (types Hantaan, Puumala, Seoul,Saaremaa, Dobrava, Sin Nombre, Andes)*Herpes simplex virus types 1 and 2 (HSV-1/2)Human herpes virus type 6 (HHV-6)HIV-1 and -2*Influenza virus types A and BJapanese encephalitis virus (JEV)*Measles virusMumps virusParainfluenza viruses types 1-4Respiratory syncytial virus (RSV)Rubella virus*Sandfly fever virus*(types Sicilian, Naples, Toscana, Cyprus)SARS Coronavirus (SARS-CoV)Tick-borne encephalitis (TBE) virusVaricella zoster virus (VZV)West Nile virus (WNV)Yellow fever virus (YFV)

Bacteria:Afipia felis*Bartonella henselaeBartonella quintanaBordetella parapertussisBordetella pertussisBorrelia afzeliiBorrelia burgdorferiBorrelia gariniiCampylobacter coli*Campylobacter jejuni*Chlamydia pneumoniaeChlamydia psittaciChlamydia trachomatisHaemophilus influenzae*Helicobacter pyloriKlebsiella pneumoniae*Legionella bozemanii*Legionella dumoffii*Legionella gormanii*Legionella jordanis*Legionella micdadei*Legionella pneumophila serotypes 1-14Listeria monocytogenes 1/2 a, 4b*Mycoplasma hominisMycoplasma pneumoniaeTreponema pallidumTreponema phagedenisUreaplasma urealyticumYersinia enterocolitica*

Bacteria antigens (EUROPLUS):Borrelia VlsE (recombinant)Borrelia OspC

Yeasts:Candida albicansCandida glabrata*Candida krusei*Candida parapsilosis*Candida tropicalis*

Parasites:Echinococcus granulosusLeishmania donovaniPlasmodium falciparum HRP-2/MSP-2 (rec.)*Plasmodium vivax MSP/CSP (recombinant)*Toxoplasma gondii

Profiles:Accompanying hepatitis profileCentral nervous system profileExanthema profileFever profile South East AsiaFlavivirus profileGastrointestinal tract profileInfectarthritis profileInfectarthritis profile (The Tropics)Lymphadenitis profileMyocarditis profileOphthalmology profileOtitis profilePregnancy profileRespiratory tract profileSexually transmitted diseases (STD) profileTORCH profile

* Currently not available as IVD in the EU.

Special substrate combinations on request

Made in Germany

Incubation with the TITERPLANETM Technique

Reagent tray

Slide fitted with BIOCHIPs

Test principle: The test system exclusively serves for  the quantitative or qualitative in vitro determination of human antibodies  in  patient  samples.  Substrate  combinations are incubated with diluted patient samples.  In the case of positive reactions, specific antibodies of the class IgA, IgG and  IgM  will  bind  to  the  antigens.  In  a  second  step,  the attached  antibodies  are  stained  with  fluorescein-labelled anti-human antibodies and made visible with  the fluores-cence microscope.

Test performance:  EUROIMMUN  BIOCHIP  slides  are  incu-bated using the proprietary TITERPLANE™ Technique. This technique enables multiple samples to be incubated next to each other and simultaneously under  identical conditions. Results are evaluated by fluorescence microscopy. 

Reproducibility:  Inter-lot  reproducibility  was  tested  with more  than  10  different  lots.  In  quantitative  evaluation  of results, the deviation amounted to no more than ± 1 fluores-cence  intensity  level.  Intra-assay and inter-assay reproduc-ibility can therefore also be guaranteed.

Reference range: Titer 1 : < 10

Sensitivity and specificity: 

Technical data:

Antigen substrate  human granulocytes (EOH/HCHO), HEp-2 cells,  liver (primate), highly purified native MPO and PR3

Sample dilution   Serum or plasma. Qualitative: 1:10, quantitative: 1:10/100/1000 etc.  Conjugate  IgG

Test procedure  30 min (sample) / 30 min (conjugate). Room temperature. 

Microskopy  Objektive  20x,  excitation  filter:  488 nm,  colour  separator:  510 nm, blocking  filter:  520 nm,  light  source:  EUROIMMUN  LED  or  mercury vapour lamp, 100 W

Reagents  Ready for use, with the exception of the PBS-Tween buffer.

Stability  18 months from the date of manufacture at +2 °C to +8 °C. 

Kit formats  10 or 20 slides, each containing 3, 5 or 10 test fields. The kits include all necessary reagents.

Order no.  FA 1201-####-23 

Test characteristics EUROPLUS™ “Granulocyte Mosaic 23”

Version: 10/07FA_1201_D_UK_A02

Panel nIIFT

cANCAEUROPLUS

Anti-PR3Anti-PR3-hn-hr

ELISAIIFT

pANCAEUROPLUS Anti-MPO

Anti-MPO ELISA

Wegener‘s granulomatosis 59 47 (80%) 45 (76%) 43 (73%) 0 0 0

Sensitivity 59 80% 76% 73% - - -

AAV, biopsy proven 112 57 (51%) 57 (51%) 57 (51%) 51 (46%) 49 (44%) 51 (46%)

AAV outpatients 74  27 (36%) 26 (35%) 26 (35%) 17 (23%) 16 (22%) 11 (15%)

Prevalence 186 45% 45% 45% 37% 35% 33%

Rheumatoid arthritis 29 0 0 0 1 (3%) 0 1 (3%)

Blood donors 27 0 0 0 0 0 1 (4%)

Non-ANCA-associated vasculitides 53 1 (2%) 1 (2%) 0 2 (4%) 2 (4%) 1 (2%)

Specificity 109 99% 99% 100% 97% 98% 97%

Systemic lupus erythematosus 100 4 (4%) 0 0  1 (1%) 0 n. d.

Sjögren‘s syndrome 196 5 (3%) 2 (1%) 1 (1%) 0 0 n. d.

Rheumatoid arthritis 200 1 (1%) 0 0 5 (3%) 5 (3%) n. d.

Asymptomatic blood donors 199 0 0 1 (1%) 0 0 n. d.

Specificity 804 99% 99% 99% 99% 99% -

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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

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

EUROIMMUN Microplate ELISA

Autoantibody determination:AMA M2-3E (IgG)ANCA Profile (IgG)ANA Screen (IgG)ANA Screen 9* or 11* (IgG)ANA VarioProfile (IgG)BP180-4X (IgG)C1q (IgG)cardiolipin (IgA, IgG, IgM, IgAGM)circulating immune complexes (CIC)cyclic citrullinated peptide (CCP; IgG)centromere protein B (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)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:GAF-3X (IgA, IgG)gliadin (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-PR3-hn-hr ELISA (IgG)

Indication: Test system for the in vitro determination of antibodies against proteinase 3 in human serum or plasma for the diagnosis of the following disease: Wegener’s granulomatosis.

Clinical significance:  ANCA  are  autoantibodies  directed  against  antigens  found  in  cytoplasmic granules of neutrophils and monocytes. Several methods are used  for  the detection of ANCA. Standard  technique  is  the  indirect  immunofluorescence  (IIF)  on  ethanol-fixed  granulocytes.  At least  two  different  staining  patterns  can  be  differentiated:  a  granular  fluorescence  which  is distributed regularly over  the entire cytoplasm of  the granulocytes,  leaving  the cell nuclei  free (cANCA:  cytoplasmic  pattern),  and  a  predominantly  smooth,  partly  fine  granular  fluorescence wrapped ribbon-like around the cell nuclei of the granulocytes (pANCA: perinuclear pattern).

ANCA are typically found in Wegener’s granulomatosis (WG), microscopic polyangiitis (MPA) in-cluding renal limited vasculitis, and Churg-Strauss syndrome (CSS), which are all forms of small-vessel  vasculitis.  These  three  diseases  are  grouped  together  as  ANCA-associated  vasculitides (AAV) according to the widely accepted classification system introduced by the Chapel Hill Con-sensus Conference. Classical cANCA are present  in most patients with WG (more than 90% in general WG with glomerulonephritis, 70% in limited WG without glomerular involvement) and in about 30% of patients with MPA. Classical cANCA are almost always directed against proteinase 3 (PR3) and very rarely against myeloperoxidase (MPO) or against PR3 and MPO simultaneously. Some  cANCA  exhibit  a  flat  homogenous  cytoplasmic  staining  in  IIF  (mostly  termed  atypical cANCA) which is often directed against bactericidal/permeability increasing protein (BPI). 

The  most  important  clinical  symptoms  of  ANCA-associated  vasculitides  are  caused  by  poor blood  supply  to  organs  or  formation  of  aneurysms  and  bleeding  due  to  destruction  of  blood vessels. Wegener’s granulomatosis  is a  febrile,  chronic granulomatosis disease, mainly of  the nasopharynx, lungs and kidney. Since cANCA have been investigated, the diagnosis of Wegen-er’s granulomatosis has tripled. Due to the high specificity of cANCA the number of diagnosed early-stage and abortive cases of Wegener’s granulomatosis increases steadily. Application of the Anti-PR3-hn-hr ELISA:  The  reagent  wells  of  the  Anti-PR3-hn-hr  ELISA  are coated with a mixture of recombinant PR3 (based on human cDNA, expressed in human cells; Sun, Specks et al., 1998) and native PR3. Owing to this, the test has a significantly higher sensi-tivity (94%) at a very good specificity (99%) compared to other ELISA only using a native antigen (88%  and  78%,  respectively;  study  performed  in  cooperation  with  the  ANCA  reference  centre University of Maastricht, Prof. Cohen-Tervaert). The International Consensus Statement recom-mends screening for ANCA using IIF and the confirmation of IIF-positive sera with both Anti-PR3 and Anti-MPO ELISA since  the combination of both  test  systems yields  the highest  specificity and sensitivity for the diagnosis of small vessel vasculitis. 

PR3

PR3

PR3

PR3

PR3

PR3

PR3

PR3

Panels (source: ANCA reference centre University of Maastricht) nAnti-PR3-hn-hr ELISA

positiveAnti-PR3 Capture

ELISA positiveAnti-PR3 ELISA

positive

AAV (cANCA- positive)

Biopsy-proven AAV 58 55 (95%) 53 (91%) 53 (91%)

AAV outpatients 35 33 (94%) 32 (91%) 26 (74%)

AAV relapses 23 23 (100%) 23 (100%) 18 (78%)

Wegener’s granulomatosis 47 43 (91%) 36 (77%) 30 (64%)

Sensitivity with respect to IIFT (cANCA) 163 154 (94%) 144 (88%) 127 (78%)

Non-ANCA-associated vasculitides (e.g. cryoglobulinemia, Henoch-Schönlein purpura, large vessel vasculitides)

55 0 2 (4%) 0

Rheumatoid arthritis 230 0 7 (3%) 0

Systemic lupus erythematosus 100 0 0 0

Sjögren‘s syndrome 200 1 (1%) 5 (3%) 2 (1%)

Blood donors, asymptomatic 429 4 (1%) 10 (2%) 3 (1%)

Specificity 1014 5 (99%) 24 (98%) 5 (99%)

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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 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: amphiphysin,CV2/CRMP5, PNMA2 (Ma-2), 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:liver-specific antigens (+ recomb. SLA/LP)neuronal antigens (+ recomb. Hu, Yo, Ri)HEp-2 cell antigens (+ SS-A and Ro-52, CENP B)Myositis ag (Mi-2, Ku, PM-Scl, Jo-1, PL-7, PL-12)

Infectious serology:

EUROLINE:EBV Profile (IgG, IgM, VCA gp125, VCA p19and EBNA-1, p22, EA-D)TORCH Profile* (T. gond., rubella, CMV, HSV-1, -2)Malaria Profile 1: Plasmodium falciparum HRP-2and MSP-2, Plasmodium vivax MSP and CSP

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

Autoantibody determination:

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 triiodothyronin (FT3)freies thyroxin (FT4)thyrotropin (TSH)calcitonin

* Currently not available as IVD in the EU.

Made in Germany

Linearity: The linearity of the Anti-PR3-hn-hr ELISA (IgG) 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-PR3-hn-hr ELISA (IgG) is linear at least in the tested concentration range of 28 RU/ml to 197 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 determinations performed in 6 different test runs.

Clinical sensitivity and specificity: Sera  from 163 patients with ANCA-associated vasculitides (AAV;  cANCA-positive),  a  control  panel  of  585  patients  with  other  diseases  and  429  healthy blood donors were analysed using the EUROIMMUN Anti-PR3-hn-hr ELISA (IgG). The sensitiv-ity of ELISA for cANCA-positive AAV was 94%, at a specificity of 99%.

Reference range:  Levels  of  anti-PR3  antibodies  were  investigated  in  429  sera  from  healthy blood donors between 19 and 68 years of age (172 women, 257 men) using the EUROIMMUN ELISA. No differences with respect to age or gender were observed. The mean concentration of antibodies against PR3 was 2.2 RU/ml  (± 9.6 RU/ml of  standard deviation) and  the values ranged  from  0.1  to  171.7  RU/ml.  With  a  cut-off  of  20  RU/ml,  4  blood  donors  were  anti-PR3 positive.

ROC analysis: In  an  analysis  of  140  samples  from  patients with  ANCA-associated  vasculitides  (cANCA-positive)  and 1014  control  samples  the  following  characteristics  were  de-termined:

Technical data:

Antigen  Mixture  of  native  proteinase  3  from  human  neutrophils  and  recom-binant proteinase 3, based on human cDNA and expressed in human cells.

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

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

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

Kit format  96 single break-off wells, incl. all necessary reagents.

Order no.  EA 1201-9601-2 G

Test characteristics Anti-PR3-hn-hr ELISA (IgG)

Version: 05/07EA_1201_D_UK_B03

Intra-assay variation, n = 20

Inter-assay variation, n = 4 x 6

SerumMean value

(RU/ml)CV (%)

Mean value (RU/ml)

CV (%)

1 55 4.1 47 11.2

2 89 2.6 85 4.3

3 108 1.8 106 4.2

4 152 2.8 159 3.9

RU/ml0.1 1 10 100 1000

Freq

uen

cyn

0

20

40

60

80

100429 Blood donors

Cut-off Specificity Sensitivity

4.9 RU/ml 95% 96%

12.0 RU/ml 98% 95%

17.6 RU/ml 99% 94%

Blood donors, n = 429

Percentile 95th 98th 99th

Cut-off 4.4 RU/ml 12.5 RU/ml 17.7 RU/ml

RU/ml0,1 1 10 100 1000

Freq

uen

cy n

0

20

40

60

80

100163 AAV patients

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

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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.

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

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 a

lanin

e

(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%

1 Sun 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)

EA_1201_I_UK_D01, 04/2008

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EUROIMMUN M e d i z i n i s c h e L a b o r d i a g n o s t i k a A G

EUROIMMUN AG · Seekamp 31 · 23560 Lübeck · Tel 0451/58 55-0 · Fax 58 55-591 · E-mail [email protected] · www.euroimmun.de

Important information for users of the EUROIMMUN Anti-PR3-hn-hr ELISA The Anti-PR3-hn-hr ELISA is a new milestone in the serological diagnosis of Wegener’s granulomatosis. The antigen substrate used consists of two components: human native proteinase 3 (PR3) and recombinant PR3 expressed in a human cell line (source: Institute for Experimental Immunology, a facility of EUROIMMUN AG). The already potent recombinant PR3 is supplemented with native antigen in order to be able to present the complete antigen spectrum, since the recombinant antigen does not always possess all native antigen epitopes. Special characteristics of the recombinant antigen component

• Human cells are used for the first time worldwide for the expression of PR3 in full-scale production. In human systems the

posttranslational modifications that take place are authentic and true to species, in contrast to (heterological) insect cells or bacteria usually used. Therefore, the target antigen used for diagnostics conforms better to the natural one.

• In recombinant PR3 the proteolytic active centre is artificially switched off by the exchange of an amino acid. Since the proteinase activity of the enzyme no longer interferes with cell metabolism, the cultured cells can accumulate PR3 in

high concentrations – without this manipulation they die early. The synthesised PR3 does not digest itself in an uncontrolled manner in the preparation and can be produced in large

quantities.

Comparison of results

The new Anti-PR3-hn-hr ELISA is much more sensitive than conventional Anti-PR3 tests: in a panel from Damoiseaux et al. (Ann Rheum Dis, 2008 Mar 28, Epub ahead of print) a sensitivity of 95% was obtained for the Anti-PR3-hn-hr ELISA (conventional Anti-PR3 ELISA: 80%), with respect to the indirect immunofluorescence test and calculated for a specificity of 99%. Four examples

Consequences for the laboratory daily routine

If conventional Anti-PR3 tests are replaced by the new Anti-PR3-hn-hr ELISA, it must be expected that: • some previously negative sera react positively (example 1), • higher measurement values are generally obtained for antibody concentrations (sometimes many times higher, examples 2 and 3), • in individual cases comparable values are obtained (example 4), • measurement values do not always increase by the same ratio; the percentage increase in values can vary substantially from serum

to serum. We recommend that you inform your customers about the change of test in writing. We are happy to provide data sheets for this purpose. If you wish you can use the following text, in whole or in part: “We will in the future be using a new test system for the determination of antibodies against PR3 (EUROIMMUN Anti-PR3-hn-hr ELISA), which detects up to 15% more cases of Wegener’s granulomatosis or ANCA-associated vasculitis. With this test a purely technically caused increase in measurement values for antibody concentrations can occur, which should not be interpreted as an increase in clinical activity. The increase can vary in strength from case to case”.

Measurement values for the concentration of Anti-PR3

AAb (IgG)

Serum

Conventional

Anti-PR3 ELISA Anti-PR3-hn-hr ELISA

1 8 RU/ml 165 RU/ml

2 22 RU/ml 91 RU/ml

3 64 RU/ml 200 RU/ml

4 196 RU/ml 200 RU/ml

EA_1201_I_UK_E01, 06/2008