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EUROIMMUN
Medizinische Labordiagnostika AG EUROIMMUN Blot Systems for Borreliosis Diagnostics: Refined and Unparalleled • Certified according to EN ISO 9001 / EN 46001 / EN ISO 13485 • In line with all recommendations of the German Society for Hygiene and Microbiology • Borrelia whole antigen extract – up to 23% more sensitive than mixes of recombinant antigens • Newly identified major Borrelia antigen VlsE in addition: a further 20% increase in sensitivity • Evaluation at a glance: anti-Borrelia IgG and IgM • EUROIMMUN: Experts in borreliosis serology! Highly differential product spectrum, including ELISA, IIFT, Westernblot and EUROLINE-WB, antigen substrates for all relevant Borrelia species available EUROIMMUN Medizinische Labordiagnostika AG Features of conventional EUROIMMUN anti-Borrelia Westernblots Monoclonal reference ab Specific antigens Monoclonal reference ab Specific antigens Monoclonal reference ab Specific antigens L83/100 22G3 p83 L83/100 22G3 p83 L83/100 22G3 p83 L75 3G10 p75 L60 E6 p60 MAB956 L39 B1 p41, Flag. MAB956 L39 B1 p41, Flag. p39, BmpA L41 1C11 L39 B5 p41, Flag. p39, BmpA p39, BmpA L34 1G8 L32 1F11 p34, OspB p31, OspA L30 1B10 L32 1F11 p30 L31 1F11 L30 1B10 p31, OspA p29, OspA p30 L22 1F8 p25, OspC p21/22 L22 1F8 p25, OspC L22 1F8 p25, OspC MAB 962 MAB 962 p21/22 MAB 962 p21 L19 A11 p19 L19 A11 p19 Borrelia burgdorferi Borrelia garinii Borrelia afzelii What role do Westernblots have in borreliosis diagnostics? Westernblots allow the serological determination of antibodies against human pathogenic Borrelia species. These include B. burgdorferi, B. garinii and B. afzelii. The German Society for Hygiene and Microbiology (Deutsche Gesellschaft für Hygiene und Mikrobiologie, DGHM), the Robert Koch Institute (Berlin) and the Center for Disease Control (Atlanta, USA) recommend a two-step strategy for the serological diagnosis of Lyme borreliosis: first a screening test – ELISA or indirect immunofluorescence (IIFT) – is employed, which must provide high sensitivity. To check the specificity, positive and borderline reactions are then analysed by Westernblot. In fresh infections we recommend performing ELISA/IIFT and Westernblot in parallel, since some weak reactions become detectable earlier in Westernblot than in the screening tests. Quality features of conventional EUROIMMUN anti-Borrelia Westernblots EUROIMMUN Westernblots fulfil near to all requirements of the German Society for Hygiene and Microbiology (Wilske et al., Lyme Borreliosis: Quality Standards for the Microbiological Diagnosis of Infectious Diseases (MiQ 12/2000): • Borrelia whole antigen extract – prerequisite for early identification of fresh infections • Clear separation of specific and unspecific antigen bands • Characterisation of all diagnostically relevant bands with monoclonal reference antibodies • Highly specific IgM detection with OspC – no false positive reactions with blood donors, as with some manufacturers’ products • Specific evaluation templates , customized to each individual test kit • Clear classification criteria for positive reactions – reaction of at least one (IgM) or at least two (IgG) specific bands • Positive, borderline and negative control sera available on request Whole antigen extract or recombinant antigens? Westernblot systems with exclusively recombinant antigens are easier to read and have led some clinicians to abandon whole antigen Westernblots. But recombinant Westernblots have a 23% lower sensitivity (Wilske et al., Med. Microbiol. Immunol. 188: 139-144, 1999), particularly with fresh infections, and therefore are not recommended for reliable borreliosis diagnostics. Individual recombinant antigens can, however, supplement whole antigen Westernblots. Additional use of the newly identified major Borrelia antigen VlsE places the whole antigen Westernblot once again at the forefront, including with regard to convenient evaluation. S n W W a R W 1 E m 6 3 1 N ( I 4 5 4 L b 3 1 0 9 C g 2 1 2 2 1 Recombinant antigens used: p83/100, p39, p41i, OspC, Osp17, p58 2 118 blood donors, 11 syphilis patients, 10 patients seropositive for rheumatoid factors EUROIMMUN AG · D-23560 Lübeck (Germany) · Seekamp 31 · Tel: +49 4509 87430 · Fax 874334 · E-mail: euroimmun@euroimmun.de L34 1G8 EUROIMMUN Medizinische Labordiagnostika AG The newly identified VlsE – major antigen for Borrelia serology What is VlsE and what is its function? VlsE ( v ariable major protein- l ike s equence, e xpressed) is a newly characterized surface protein of Borrelia burgdorferi . Its significance for the serological diagnosis of borreliosis was until recently overlooked. VlsE plays a key role in the survival strategy of Borrelia. After penetration into the host organism, Borrelia bacteria constantly change their surface-expressed VlsE and, in this way, try to escape recognition and elimination by the immune system. The VlsE protein is divided into several sections: conserved regions, which serve as trans- membrane domains and anchor VlsE in the bacterial membrane, as well as variable and invariable regions. The variable regions of VlsE face outwards and are constantly changed by recombination (see below), whereby the attacking immune system consistently encounters new, altered antigen epitopes. The invariable regions are masked by the variable regions and, in living Borrelia bacteria, are protected from direct attack by the immune system. When deceased Borrelia bacteria are processed by antigen-presenting cells, the complete VlsE protein is presented to the immune system and the host also forms antibodies against the invariable and conserved regions of VlsE . These antibodies cannot bind to the Borrelia bacteria in vivo, since the specific epitopes are masked, but they are highly suitable for diagnosis of borreliosis because of the high level of conservation of their target antigens. Using enzyme immunoassays (ELISA, EUROLINE-WB) Lyme borreliosis can be diagnosed in 85% of cases, regardless of the species, through the detection of antibodies against VlsE alone. invariable regions variable regions outer Borrelia membrane inner conserved regions VlsE on the surface of Borrelia How is the variability of the VlsE variable regions generated? For production of the VlsE protein, the DNA of Borrelia bacteria contains 15 to 20 so-called v ariable major protein- l ike s equence( vls ) cassettes, which act as a library of genetic information. They are each composed of 12 gene sections: six invariable regions (red) and six variable regions (green). By combining different elements from these cassettes (so-called recombination), an almost unlimited number of surface proteins differing in their variable regions can be produced. The DNA which is assembled from the individual cassettes and used for synthesis of the protein is called vlsE (E = expressed). It also contains the transmembrane domains of the VlsE protein. VlsE is expressed exclusively in vivo; cultured Borrelia do not contain this antigen. Literature: Lawrenz et al., J. Clin. Microbiol. 37: 3997-4004 (1999); Eicken et al., J. Biol. Chem. 277: 21691-21696 (2002) vls cassette 4 vls cassette 5 vls cassette 6 vls cassettes of Borrelia DNA (section) Combination of individual cassette elements vlsE DNA Expression of VlsE protein variable regions (VR) VR VR VR VR VR VR VlsE protein IR IR IR IR IR IR invariable regions (IR) conserved regions (transmembrane domains) Recombination and expression of VlsE EUROIMMUN AG · D-23560 Lübeck (Germany) · Seekamp 31 · Tel: +49 4509 87430 · Fax 874334 · E-mail: euroimmun@euroimmun.de EUROIMMUN Medizinische Labordiagnostika AG Anti-Borrelia EUROLINE-WB – optimal borreliosis diagnostics with Borrelia whole antigen extract plus VlsE IgM Rapid evaluation IgG Anti-Borrelia EUROLINE-WB L60 E6 p17 p19 p21 p25, OspC p30 p31, OspA p39, BmpA p41, Flag. p83 VlsE Anti-Borrelia EUROLINE-WB EUROLINE-WB – a significant advance in antibody diagnostics In the line blot system EUROLINE , purified, biochemically characterized antigens are printed as parallel lines at defined positions on the membrane. Reactions can be evaluated effortlessly, but the spectrum of available antigens is often limited. Conventional native Western- blot systems provide the complete antigen spectrum of cultured cells, tissue or infectious agents, whereby the individual proteins are separated electrophoretically according to size. However, the multitude of antigens on the membrane strips makes the evaluation more difficult, since the antigen bands often lie very close to one another. The EUROLINE-WB combines the advantages of both methods: Ready prepared Westernblot strips are fitted with EUROLINE membrane chips , which are preprinted with either native, affinity chromatographically purified antigens or recombinant antigens. For each antigen the most suitable membrane is selected and the optimal coating procedure used. Why is VlsE additionally employed as a recombinant antigen? A study by the Max von Pettenkofer Institute (German Borrelia Reference Centre) showed that by additionally determining antibodies against VlsE the serological hit rate can be increased by 20% compared to whole extract Westernblots and by 30% compared to recombinant antigen Westernblots. Of all recombinant antigens tested, VlsE possesses the highest sensitivity for the detection of a Borrelia infection (Schulte-Spechtel et al., J. Clin. Microbiol. 41:1299-1303, 2003).These results could be confirmed in an internal study (Meyer et al., scientific presentation submitted to the 32 nd Congress of the German Society for Rheumatology, Frankfurt, 2003). Over 85% of IgG-positive sera could be identified at a glance by assessing the VlsE band. VlsE allows detection of antibodies against all Borrelia species, and the risk of a false negative reaction due to species differences is ten times lower. A e P r i n b p ( = 3 R V 8 W a e 6 R a m 5 p 0 p p O O p Quality features of the EUROIMMUN anti-Borrelia EUROLINE-WB (whole antigen plus VlsE) E e w c s v Performance of test with B w a e h s v i f f i IgM conjugate IgG conjugate V a h s v f a s H s I r w O h b d s n v OspC band stained Neither VlsE nor OspC band stained VlsE band stained I o 8 I v s a a g ( I o a p v r ( a s anti-Borrelia IgM positive Evaluation of other bands anti-Borrelia IgG positive Q l w D r Evaluation of anti-Borrelia EUROLINE-WB The following EUROIMMUN test systems contain the major Borrelia antigen VlsE: Anti-VlsE ELISA (Order no.: EI 2132-9601-1 G or M) and Anti-Borrelia EUROLINE-WB (Order no.: DY 2131-1601-1 G or M) . The evaluation system EUROLineScan , developed by EUROIMMUN, provides fully automated evaluation of Westernblot strips and documentation of results. EUROIMMUN AG · D-23560 Lübeck (Germany) · Seekamp 31 · Tel: +49 4509 87430 · Fax 874334 · E-mail: euroimmun@euroimmun.de DY_2131_I_GB_A01 i |
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