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 +49 451 58550 · Fax 5855591 · E-mail euroimmun@euroimmun.de MV_5110_I_UK_A02, 04/2009

HLA-B27 determination using EUROArrayA molecular genetic microarray test system for the diagnosis

of ankylosing spondylitis

Easy test performance due to prefabricated PCR components and known incuba-tion technique

Simple protocol – expert knowledge in molecular biology is not required

Fully automated unbiased evaluation and diagnosis using EUROArrayScan

Highest sensitivity: all known HLA-B27 alleles are detected

Improved specifi city: non-disease-associated HLA-B27 alleles are detected

Many integrated control mechanisms document correct test performance

Test system, scanner and software are validated in accordance with IVD guide-lines

In contrast to cytotoxicity tests, blood samples can be collected and processed together, e.g. once per week; the test performance can be interrupted at different steps and resumed the next working day

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Order entry and

automatic protocol creation

(EUROArrayScan software)

DNA extraction from EDTA blood

PCR amplifi cation using HLA-B27

specifi c primers

Microarray hybridisation

Scanning of reactions using

Microarray scanner

Data analysis and creation of

result report (EUROArrayScan

software)

HLA-B27 Microarray test system EUROIMMUN Microarray scanner

Evaluation using EUROArrayScan Protocol for each individual reportTest performance

1 mm

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  ·  23560  Luebeck  (Germany)  ·  Seekamp  31  ·  Telephone  +49  451  58550  ·  Fax  5855591  ·  E-mail  euroimmun@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

Indication: Test system for the determination of disease-associated HLA-B*27 alleles in human, genomic DNA for the diagnosis of the following diseases: Bechterew’s disease (spondylitis anky-losans, ankylosing spondylitis, AS), Reiter’s disease (combination of symptoms from urethritis, conjunctivitis/uveitis, arthritis), reactive arthritis (para-/postinfectious arthritis), acute uveitis an-terior or acute iridocyclitis, periarthritis (periarthropathia) humeroscapularis, arthritis psoriatica (psoriasis arthritis), juvenile idiopathic arthritis, enteropathies (chronic-inflammatory bowel dis-eases).

Clinical significance: Human leukocyte antigens (HLA) are tissue antigens (membrane-associated glycoproteins) of the human major histocompatibility complex (MHC). HLA-B belongs to the HLA antigens of class I (also called MHC I antigens) which are present in all nucleus-containing body cells. Their function is the control of the T-cell-mediated immune response. Due to an extreme genetic polymorphism there are a large number of HLA phenotypes. For HLA-B over 1000 differ-ent alleles have been described. The HLA-B*27 allele alone can be divided into 51 subtypes, which differ only in a few bases (B*2701 to B*2743). 

The membrane protein HLA-B27 is associated with the occurrence of several autoimmune dis-eases. In Western Europe the prevalence of HLA-B27 is around 6 to 9%. When HLA-B27 is present, the relative risk of Bechterew’s disease (ankylosing spondylitis) is 87.4, for Reiter’s disease (symp-tom combination urethritis, conjuncativitiy(uveitis, arthritis) 37, for reactive arthritis (para-/post-infection arthritis) 14 to 21 (depending on the infectious agent), for acute uveitis anterior or acute iridocyclitis 10.4, for periarthritis (periarthropathy) humeroscapularis 6.3, for arthritis psoriatica (psoriasis arthritis) 4 and for juvenile idiopathic arthritis 3.2. Enteropathies (chronic-inflammatory bowel diseases) are also associated with HLA-B27.

Bechterew’s disease  is a chronic  inflammatory  rheumatic disease of  the axial  skeleton  (spinal column, iliosacral joints, pubic symphysis and facet joints), the extremity joints and tendon inser-tions. It affects mainly men between the ages of 15 and 30. There is a clear relationship between the disease and the presence of HLA-B27. Around 90% of Bechterew’s disease patients are car-riers of this tissue antigen, in particular subtypes B*2702, B*2704 and B*2705. Subtypes B*2706 and B*2709 are, on the other hand, not associated with this disease. HLA-B*27 determination is therefore important for differential diagnostics.

Application of the EUROArray HLA-B27:  HLA-B27  can  be  determined  accurately  and  precisely with molecular biological methods via  the detection of  the corresponding allele  (HLA-B*27)  in the genomic DNA of the patient. The method competes with the lymphocytotoxicitiy tests used up until now. In particular, PCR (polymerase chain reaction) using allele-specific primers has the potential  to  deliver  reliable  results,  especially  for  the  various  HLA-B*27  subtypes.  Because  of cross reactions with antibodies (with e.g. HLA-B7) and potential false-negative results in immu-nophenotyping when HLA-B*27 expression is low, molecular genetic determination of HLA-B*27 is more specific and sensitive  than serological methods.  In  this EUROIMMUN  test  system  the HLA-B*27 primers are chosen and optimised so that all currently known HLA-B*27 subtypes are detected. Furthermore, when a positive result is obtained, it is indicated whether subtypes HLA-B*2706 or HLA-B*2709 could be involved. These two subtypes are not associated with the occur-rence of ankylosing spondylitis.

EUROArray HLA-B27 Molecular genetic microarray test system

Evaluation using EUROArrayScan Protocol for each individual report

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 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

EUROIMMU AG  ·  23560  Luebeck  (Germany)  ·  Seekamp  31  ·  Telephone  +49  451  58550  ·  Fax  5855591  ·  E-mail  euroimmun@euroimmun.de

Test principle: The EUROArray is an in vitro test for molec-ular genetic determination of disease-associated HLA-B*27 alleles  in human, genomic DNA.  In the first reaction step, two sections  (exon 2 and exon 3) of  the HLA-B gene and a ß-globin gene fragment as positive control are amplified from a genomic patient DNA sample using the polymerase chain  reaction  (PCR).  The  HLA-B  gene  sections  are  only amplified  if  the  sample  contains  an  HLA-B*27  allele.  All PCR products are labelled with a fluorescence dye as they are produced. In the second reaction step, the products are analysed  using  the  microarray,  which  contains  immobi-lised probes that are complementary to the amplified DNA. The specific binding (hybridisation) of the fluorescing PCR product  to  the corresponding microarray spot  is detected using a EUROIMMUN microarray scanner. A fluorescence signal  on  the  HLA-B*27-specific  spots  indicates  the  pres-ence of an HLA-B*27 allele in the patient DNA sample. 

Test procedure: The PCRs are first incubated in the thermo-cycler and then, using the TITERPLANE™ technique, on EUROArray slides containing microar-ray BIOCHIPs. Scanning and evaluation are performed using the EUROIMMUN Microarray scan-ner and the EUROArrayScan programme. The programme enables fully automated evaluation of EUROArray analyses and detailed documentation of results. 

Sensitivity and specificity: The microarray test system detects all HLA-B*27 subtypes and indicates the possible presence of the non-disease associated subtypes HLA-B*2706 and HLA-B*2709.

Robustness: A total of 106 DNA samples from blood donors were investigated simultaneously in one test run. All determinations were successful.

Prevalence: In the investigation of 106 randomly selected samples 9.4% of cases were positive. This  value  corresponds  to  the  known  prevalence  of  the  marker  in  Central  European  popula-tion.

Technical data:

Substrate  Single-stranded DNA probes, length: 20 to 50 nucleotides.

Test procedure  60 min (PCR) / 60 min (hybridisation) / 5 min (automated evaluation).

Reagents  Ready for use.

Controls  Integrated HLA-B27 positive control (no additional positive control re-quired).  Integrated DNA positive control.

CE-IVD certified  Complete process incl. DNA extraction is validated.

Test kit format  20 slides, each containing 5 test fields. 

Order number  MN 5110-2005

Test characteristics Molecular genetic microarray test system

Reference samples nReference method

Sensitivity Specificity

EDTA blood samples from patients and blood donors, Germany(15 precharacterised as HLA-B*27 positive, 48 as HLA-B*27 negative)

63molecular 

genetic100 % 100 %

DNA samples from patients and blood donors, Germany(30 precharacterised as HLA-B*27 positive, 30 as HLA-B*27 negative)

60molecular 

genetic100 % 100 %

DNA samples from the “International Histocompatibility Working Group” (IHWG): “Sequence Polymorphism Reference Panel (SP Reference Panel)” and “HLA (Anthropology) Reference Panel” (6 precharacterised as HLA-B*27 positive, 49 as HLA-B*27 negative)

55molecular 

genetic100 % 100 %

DNA sample

PCR (polymerase chain reaction)

DNA microarray hybridisation

Fully automated evaluation

Version: 05/09MV_5110_D_UK_A01