SERION ELISA classic. Epstein-Barr Virus VCA IgG/IgM, Epstein-Barr Virus EBNA1 IgG, Epstein-Barr Virus EA IgG YOUR GLOBAL PARTNER DIAGNOSTICS

Transcript

1 Manufacturer Fabricante Κατασκευαστής Fabricante Výrobce Institut Virion\Serion GmbH Friedrich-Bergius-Ring 19 D Würzburg, Germany Telefon: +49 (0) 9 31 / Fax: +49 (0) 9 31 / dialog@virion-serion.de Internet: SERION ELISA classic Epstein-Barr Virus YOUR GLOBAL PARTNER IN DIAGNOSTICS SERION ELISA classic Epstein-Barr Virus VCA IgG/IgM, Epstein-Barr Virus EBNA1 IgG, Epstein-Barr Virus EA IgG Instructions - English Instrucciones de empleo - Español Oδηγίες χρήσης - Ελληνικά Instruções de emprego - Português Pokyny - Česky (Version/Versión/Έκδοση/Versão/Verze 22.11/12-1) KAL136-2

2 Updates Please pay attention to the differences in comparison to the previous version. Current version Nr.: V 22.11/12-1 Previous version: V 21.10/01-1 Update in section: General Update, 5, SERION ELISA classic Epstein-Barr Virus VCA IgG/IgM, Epstein-Barr Virus EBNA1 IgG, Epstein-Barr Virus EA IgG CONTENTS 1 INTENDED USE 2 DIAGNOSTIC RELEVANCE EN GR ES 3 TEST PRINCIPLE SERION ELISA classic 4 KIT COMPONENTS 5 MATERIAL REQUIRED BUT NOT SUPPLIED 6 STORAGE AND STABILITY 7 TEST PROCEDURE SERION ELISA classic 7.1 Evidence of Deterioration CZ PT 7.2 Sample Preparation and Storage 7.3 Preparation of Kit Reagents 7.4 Overview - Test Procedure 7.5 Manual Test Procedure 7.6 Automated Test Procedure 7.7 Positive Control / Accuracy Control 8 TEST EVALUATION 8.1 Single-Point Quantification with the 4PL Method 8.2 Criteria of Validity 8.3 Calculation SERION ELISA classic EBV VCA IgG/IgM, EBV EBNA1 IgG, EBV EA IgG 8.4 Limits of Quantification 8.5 Borderline Ranges 8.6 Interpretation of Results 8.7 Reference Range of healthy Individuals 9 PERFORMANCE CHARACTERISTICS 9.1 Sensitivity and Specificity 9.2 Reproducibility 10 SAFETY MEASURES 10.1 Statements of Warning 10.2 Disposal 11 REFERENCES current version No.: V 22.11/12-1 previous version: V 21.10/01-1

3 Pos: 1 /Arbeitsanl eitung en ELISA classi c/gültig für nur ein Dokument/U pdate/u pdate: Pos: 6 /Arbeitsanl eitung en ELISA classi c/gültig für alle D okumente/elisa cl assic/allgemei ne T exte ELISA cl assic/kapitelüberschrift "Diag nostische Pos: 4\mod_ _18.doc 2 /Arbeitsanleitungen Texte classic/einleitung classic/gültig für "Enzymimmunoassay" alle Dokumente/ELISA SERION ELISA classic Epstein-Barr Virus VCA IgG/IgM, Epstein-Barr Virus EBNA1 IgG, Epstein-Barr Virus EA IgG Enzyme-immunoassay for determination of human antibodies for in vitro diagnostic use Pos: ELISA Bestellnummern classic/gültig nur 0\mod_ _18.doc ein SERION ELISA classic Epstein-Barr Virus VCA IgG Order Nr.: ESR1361G SERION ELISA classic Epstein-Barr Virus VCA IgM Order Nr.: ESR1361M SERION ELISA classic Epstein-Barr Virus EBNA1 IgG Order Nr.: ESR1362G SERION ELISA classic Epstein-Barr Virus EA IgG Order Nr.: ESR1363G Pos: classic/allgemeine 0\mod_ _18.doc 4 /Arbeitsanleitungen Texte 170 classic/kapitelüberschrift für alle Dokumente/ELISA 1 INTENDED USE Pos: 5 /Arbeitsanl eitung en ELISA classi c/gültig für nur ein Dokument/Anwendungsber eich/ebv: SERION ELISA classic Epstein-Barr Virus EA IgG, VCA IgG, IgM and EBNA1 IgG tests are quantitative and qualitative immunoassays for the detection of human antibodies in serum or plasma directed against components of the Epstein-Barr Virus (EBV). The SERION ELISA classic Epstein-Barr Virus EA IgG and SERION ELISA classic EBV VCA IgM are recommended for the detection of acute infections or reactivations. The SERION ELISA classic Epstein-Barr Virus VCA IgG determines primary and recent infections. The SERION ELISA classic EBNA1 IgG is recommended for the determination of past infections. 2 DIAGNOSTIC RELEVANCE Pos: 7 /Arbeitsanl eitung en ELISA classi c/gültig für nur ein Dokument/Di agnostische Bedeutung/EBV: Diagnostische The Epstein - Barr virus (EBV) is a human pathogenic virus belonging to the Herpesvirus family. It occurs worldwide and, in common with all Herpesviruses, the prevalence in the general population is high, reaching 90 to 95 % in the adult population. In the so called developing countries, primary infection is generally in the first year of life and is often asymptomatic. In contrast, in countries with high standards of hygiene primary infection occurs mainly in teenagers and young adults. The peak age for infection is between 15 and 20 years and around 50 % of those infected will develop an infectious mononucleosis. Transmission is primarily through exchange of saliva although other routes are possible such as from blood products or bone marrow transplantation. During a primary infection the salivary glands are initially involved and virus reaches the nose and throat via the saliva. At this stage, the symptoms of infection are flu-like. The virus is disseminated throughout the body by infected B lymphocytes, which are normally regulated by the immune system but are stimulated to proliferate by the virus. These infected B lymphocytes in the peripheral blood are characteristically atypical with a variable shape, distinctly basophile cytoplasm and an obvious nucleus. As the disease progresses it may manifest as high fever, splenomegaly, lymphadenitis, thrombocytopenia, and hepatitis. Due to the virus s dissemination and transmission strategy, primarily via saliva, infectious mononucleosis (IM) or glandular fever has also been called the kissing disease. In rare cases an acute IM infection can lead to a chronic active disease state. In such cases the symptoms of IM may continue for a considerable time period. The pathogenesis english 2

4 of this complication is unclear although genetic predisposition and/or infection with a particularly lytic strain of virus are suspected. EN Another rare disease state is chronic IM which, in contrast to chronic acute infection, following resolution of symptoms and a period of latency, possibly lasting several years, reactivates with sometimes fatal consequences. The reasons and causes behind such cases are unclear. Some genetic disorders such as XLPS (x-linked lymphoproliferative syndrome) can result in uncontrolled B cell proliferation following infection with EBV and frequently lead to chronic infection. Medical suppression of the immune system such as used in transplant patients may lead to the reactivation of latent virus or a increased likelihood of primary infection can lead to an EBV induced transplant rejection. AIDS patients are also, as a consequence of their underlying immune disease, more susceptible to infection and reactivations. In certain geographical regions EBV is closely associated with the incidence of nasopharyngeal carcinoma. This carcinoma of the pharynx, nose and throat consists of undifferentiated epithelial cells and has a propensity for metastasis. This disease occurs globally but is particularly common in certain regions of south China. Genetic predisposition as well as environmental aspects such as diet are under discussion as cofactors. Burkitt s lymphoma (BL) is also a tumor associated with EBV, primarily in the geographical regions of Africa and Papua New Guinea. This monoclonal B-cell tumor is also linked to areas with high incidence of malaria and over 90 % of such tumors show evidence of the E-B virus. The influence of plasmodium infection on the immune response brings the role of malaria as a cofactor for BL into discussion. Sporadic cases of BL in other regions do occur, however in such cases EBV is much less commonly detected and other cofactors such as genetic changes through chromosome translocation are thought to be responsible for the tumour. The antibody response to EBV infection is extremely variable as a consequence of the complexity of the virus and the heterogeneous of the various stages of infection. During the active phase of infection, antibodies directed at approximately 100 different viral antigens are produced. This drops to around 10 during latent infection (EBNA 1-6, Late Membrane Proteins 1-3). With this background there has been a trend towards the development of antibody detection systems which utilise single components / proteins rather than the complete virus. In this way it is possible to correlate the production of antibodies to certain antigens with the different disease phases and disease progression. english 3

5 Pos: 8 /Arbeitsanl eitung en ELISA classi c/gültig für alle D okumente/elisa cl assic/t estprinzip/testpri nzip ELISA Symptoms Heterophile ab VCA-IgM weeks months years Figure 1: Antibody titers after EBV-infection. Source: Figure 1 shows the typical immune response in terms of antibody production following infection with EBV. Very early in infection IgM is produced against the early antigen (EA). Maximum concentrations of antibodies tend to coincide with the onset of symptoms, and approximately two weeks after initial infection EA IgG, VCA (Virus Capsid Antigen) IgM and VCA IgG production begins. The highest anti-vca IgM concentrations are found around three weeks following the onset of symptoms. EA IgG are often detectable for a considerable time following infection. Subsequently the concentrations of VCA IgM and later EA IgG antibodies fall. Anti VCA IgG reaches a peak some six weeks after symptoms appear and remain at a high level lifelong. Around three weeks after appearance of symptoms EBNA1 IgG antibodies, which are an indicator of a past infection, begin to be produced and reach a peak at around seven months and remain at a high level for life following a normal EBV infection. Reactivation of the virus (such as immunosuppression) usually leads to a significant increase in EA IgG antibodies while VCA IgM titres seldom increase. Only very occasionally, following immunosuppression, does EBNA1 IgG disappear so that EA IgG is a good marker for reactivation. english 4

6 Pos: 9 /Arbeitsanl eitung en ELISA classi c/gültig für nur ein Dokument/Inhalt und Z usammensetzung/ebv: Inhalt und Z 3 TEST PRINCIPLE SERION ELISA classic EN The ELISA (Enzyme Linked Immunosorbent Assay) is an immunoassay, which is particularly suited to the determination of antibodies in the field of infectious serology. The reaction is based on the specific interaction of antibodies with their corresponding antigen. The test strips of the SERION ELISA classic microtiter plate are coated with specific antigens of the pathogen of interest. If antibodies in the patient s serum sample are present, they bind to the fixed antigen. A secondary antibody, which has been conjugated with the enzyme alkaline phosphatase, detects and binds to the immune complex. The colourless substrate p-nitrophenylphosphate is then converted into the coloured product p-nitrophenol. The signal intensity of this reaction product is proportional to the concentration of the analyte in the sample and is measured photometrically. english 5

7 4 KIT COMPONENTS Test Components Pieces / Volume Break apart microtiter test strips each with eight antigen coated single wells, (altogether 96) MTP, 1 frame. The coating material is inactivated. 12 pieces Standard serum (ready-to-use) STD, Human serum in protein containing phosphate buffer; negative for anti-hiv Ab, HBs-Ag (Hepatitis B-Virus surface antigen) and anti-hcv Ab; preservative: < 0.1 % sodium azide; colouring: Amaranth O. 2 x 2 ml Negative control serum (ready-to-use) NEG, Human serum in protein containing phosphate buffer; negative for anti-hiv Ab, HBs-Ag (Hepatitis B-Virus surface antigen) and anti-hcv Ab; preservative: < 0.1 % sodium azide; colouring: Lissamin Green V. 2 ml Anti-human IgA, IgG or IgM conjugate (ready-to-use) APC, Anti-human IgA, IgG or IgM polyclonal antibody, conjugated to alkaline phosphatase, stabilised with protein stabilisation solution; preservative: 0.01 % methylisothiazolone, 0.01 % bromnitrodioxane. 13 ml Washing solution concentrate (sufficient for 1000 ml) WASH, Sodium chloride solution with Tween 20 and 30 mm Tris/HCl, ph 7,4; preservative: < 0.1 % sodium azide. 33,3 ml Dilution buffer DILB or special dilution buffer S1 DILBS1, (Special dilution buffer for SERION ELISA classic EBV EA IgG. Shake immediately before use!) Protein containing phosphate buffer with Tween 20 (DILB) and lysate of E. coli ( DILBS1 ); preservative: < 0.1 % sodium azide; colouring: 0.01 g/l Bromphenol blue. 2 x 50 ml Stopping solution STOP, 1.2 N sodium hydroxide. 15 ml Substrate (ready-to-use) pnpp, Para-nitrophenylphosphate in solvent free buffer; preservative: < 0.1 % sodium azide (Substrate in unopened bottle may have a slightly yellow coloring, which does not reduce the quality of the product!) 13 ml Quality control certificate with standard curve and evaluation table INFO, 2 pages (quantification of antibodies in IU/ml or U/ml). Pos: benötigte Nachweis) 10 /Arbeitsanleitungen 5\mod_ _18.doc Zusätzlich ELISA classic/gültig Materialien mehrere (für Dokumente/Zusätzlich Teste mit IgM- english 6

8 5 MATERIAL REQUIRED BUT NOT SUPPLIED EN - common laboratory equipment - for the IgM detection: SERION Rf-Absorbent, order no. Z200 (20 ml) - photometer for microtitre plates with filter, wavelength 405 nm, recommended reference wavelength 620 nm nm (e.g. 650 nm) - incubator 37 C - moist chamber - distilled water - Click-Clips (order no. VT120) Pos: 11 /Arbeitsanleitungen ELISA classic/gültig für alle Dokumente/ELISA classic/lagerung und Haltbarkeit/Lagerung und english 7

9 Pos: 12 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/überschrift: Durchführ 6 STORAGE AND STABILITY Reagent Storage Stability Microtiter strips (coated with antigen) unopened see expiry date; after opening at 2 8 C in closed aluminum bag with desiccant minimum shelf-life: four weeks; Control sera / Standard sera Strips which are not used must be stored dry in the closed aluminum bag. after opening at 2 8 C shelf-life in case of proper use and storage until expiry date see expiry date; 24 months as of production Conjugate ready-to-use solution at 2 8 C Avoid contamination e.g. by using sterile tips. see expiry date; 28 months as of production Dilution buffer Unopened after opening at 2 8 C Discard cloudy solutions. see expiry date; 36 months as of production; 24 months Washing solution Concentrate after opening at 2 8 C working dilution at 2 8 C working dilution at room temperature Bottles used for the working dilution should be cleaned regularly. Discard cloudy solutions. see expiry date; 2 weeks; 1 week Substrate ready-to-use solution at 2 8 C, stored protected from light Avoid contamination e.g. by using sterile tips. Discard if solution turns yellow (extinction against aqua dest. > 0.25 OD). see expiry date; 36 months as of production Stopping solution After opening at room temperature see expiry date english 8

10 Pos: 13 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/allgemeine Hi nweise ELISA cl 7 TEST PROCEDURE SERION ELISA classic EN 7.1 Evidence of Deterioration Only use SERION ELISA classic reagents when using SERION ELISA classic immunoassays. The components must not be exchanged for reagents of other manufacturers. Standard and control sera of SERION ELISA classic immunoassays are defined exclusively for the test kit to be used and must not be used in other lots. Dilution buffer, washing solution, substrate and stop solution can be used for all SERION ELISA classic immunoassays irrespective of the lot and the test. There are three different conjugate concentrations for each immunoglobulin class: LOW, MEDIUM, HIGH. The classification is written on each label as follows: e.g. IgG + low concentrated IgG conjugate IgG ++ medium concentrated IgG conjugate IgG +++ high concentrated IgG conjugate In rare cases the use of special conjugate is necessary to guarantee consistent quality of our products. Special conjugates are produced in a separate lot and do not carry the + sign and are not exchangeable with other conjugates. Please pay close attention to information on labels! Unopened, all components of the SERION ELISA classic tests may, if stored accordingly, be used up to the expiry dates given on the labels. Reagents may not be used after date of expiry. Dilution or alteration of the reagents may result in a loss of sensitivity. Avoid exposure of reagents to strong light during storage and incubation. Reagents must be tightly closed after use to avoid evaporation and contamination. To open the aluminum bag of the microtiter plate please cut off the top of the marked side only, in order to guarantee proper reclosing. Do not use the strips if the aluminum bag is damaged or if the bag with remaining strips and desiccant was not properly reclosed. Use aseptic techniques when removing aliquots from the reagent tubes to avoid contamination. To avoid false positive results ensure not to contact or splash the top-walls of wells while pipetting conjugate. Take care not to mix the caps of the bottles and/or vials. Reproducibility of test results is dependent on thorough mixing of the reagents. Agitate the flasks containing control sera before use and also all samples after dilution (e.g. by using a vortex mixer). Be sure to pipette carefully and comply with the given incubation times and temperatures. Significant time differences between pipetting the first and last well of the microtiter plate english 9

11 Pos: 15 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/kapi tel überschrift Pos: 16 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/testdurchführ ung/probenverdünnung/ebv: Pr obenver dünnung, T eil Pos: 18 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/testdurchführ ung/probenverdünnung/ebv: Pr obenver dünnung, T eil 2 Ü when dispensing samples and control sera, conjugate or substrate can result in different pre-incubation times, which may influence the precision and reproducibility of the results. Optimum results can only be achieved if the instructions are strictly followed. The SERION ELISA classic immunoassay is only valid if the lot-specific validation criteria on the quality control certificate are fulfilled. Adequate washing avoids test unspecificities. Therefore, the washing procedure should be carried out carefully. All of the flat bottom wells should be filled with equal volumes of washing buffer. At the end of the procedure ensure that the wells are free of all washing buffer in order to avoid uncontrolled dilution effects. Avoid foaming! Take care not to damage the inscription (pathogen / antibody class) on the microtiter test strips during washing and aspiration to avoid confusion. Pos: 14 /Ar bei tsanl eitungen ELISA cl assic/gültig für mehrer e D okumente/t estdurchführ ung/probenvor ber. und Lager ung (für ALLE Err eger auß er Borreli a, CM V, FSM E, H SV,Masern,Mumps,R öteln,vz 7.2 Sample Preparation and Storage Lipaemic, hemolytic or icteric samples (serum or plasma) should only be tested with caution. Obviously contaminated samples should not be tested. Serum or plasma (EDTA, citrate, heparin) collected according to standard laboratory methods are suitable samples. Samples must not be thermally inactivated Dilution of Samples Before running the test, patient samples (V 1 ) must be diluted in dilution buffer (V 2 ) as follows: SERION ELISA classic Epstein-Barr Virus EA IgG (special dilution buffer B231-S1!) SERION ELISA classic Epstein-Barr Virus VCA IgG (dilution buffer B231) SERION ELISA classic Epstein-Barr Virus EBNA1 IgG (dilution buffer B231) Dilution buffer for SERION ELISA classic EBV EA IgG (B231-S1) is not exchangeable against the dilution buffer for SERION ELISA classic EBV VCA or EBNA1. V 1 + V 2 = add 10 µl patient s sample each to 1000 µl dilution buffer Pos: 17 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/pr obenver dünnung: Mischung der After dilution and before pipetting into the microtiter plate the samples must be mixed thoroughly to prepare a homogenous solution. english 10

12 SERION ELISA classic Epstein-Barr Virus VCA IgM EN Dokumente/Testdurchführung/Probenverdünnung: IgM-Nachweis) Pos: 19 0\mod_ _18.doc ELISA classic/gültig für mehrere (für Tests mit Interference with rheumatoid factors Rheumatoid factors are autoantibodies mainly of the IgM class, which preferably bind to IgG immune complexes. The presence of non-specific IgM antibodies (rheumatoid factors) can lead to false-positive results in the IgM assay. Furthermore, the possibility exists, that weak-binding pathogen-specific IgM antibodies may be displaced by stronger-binding IgG antibodies leading to a false negative IgM result. Therefore it is necessary to pretreat samples with rheumatoid factor-absorbens prior to IgM detection (SERION RF-Absorbent, Order Nr.: Z200 (20 ml/100 tests)). Rf-absorption is performed by incubation of the patient s sample in Rf-dilution buffer for 15 minutes at room temperature or over night at 4 C. The test procedure is described in a separate instruction manual. Pos: 20 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/testdurchführ ung/probenverdünnung/ebv: Pr obenver dünnung T eil Before running the test, rheumatoid factor-absorbent (V 1 ) must be diluted 1+4 in dilution buffer (V 2 ). V 1 + V 2 = V 3 (1 + 4) add 200 µl Rf-absorbent each to 800 µl dilution buffer Patient s samples (V 4 ) must be diluted in this Rf-dilution buffer (V 3 ): V 4 + V 3 = add 10 µl patient s sample each to 1000 µl Rf-dilution buffer Pos: 21 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/pr obenver dünnung: Mischung der After dilution and before pipetting into the microtiter plate the samples must be mixed thoroughly to prepare a homogenous solution. Pos: 22 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/pr obenlager Sample Storage The patient s samples should not be stored for more than 7 days at 2 8 C. Extended storage is possible at -20 C. Avoid repeated freezing and thawing of samples. Diluted samples can be stored at 2 8 C for one week. Pos: 23 /Arbeitsanleitungen ELISA classic/gültig für alle Dokumente/ELISA classic/testdurchführung/reagenzienvorbereitung, Teil english 11

13 Pos: 25 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/reagenzienvorberei tung, T eil Pos: 26 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/testabl auf/ü berschrift 7.3 Preparation of Kit Reagents Bring all reagents to room temperature before testing Microtiter Test Strips The microtiter test strips in frames are packed with a desiccant in an aluminum bag. Take unrequired cavities out of the frame and put them back into the aluminum bag. Close bag carefully to ensure airtight conditions Control Sera / Standard Sera Control and standard sera are ready-to-use and must not be diluted any further. For each test run - independent of the number of microtiter test strips to be used - control and standard sera must be included. The standard sera should be set up in duplicate. Pos: 24 /Ar bei tsanl eitungen ELISA cl assic/gültig für mehrer e D okumente/t estdurchführ ung/r eagenzienvorbereitung: Rf- Absorbens (für T este mit Do not treat control sera with Rf-absorbent Anti-human IgA, IgG or IgM AP-Conjugate (ready-to-use) Conjugates with the same concentration and of the same immunoglobulin class are interchangeable. Avoid contamination of ready-to-use conjugates e. g. by using sterile tips Washing Solution Dilute washing buffer concentrate (V 1 ) 1:30 with aqua dest. to a final volume of V 2. Example: Buffer concentrate (V 1 ) Final volume (V 2 ) 33.3 ml 1000 ml 1.0 ml 30 ml Dilution Buffer for Samples (ready-to-use) Special dilution buffer for SERION ELISA classic EBV EA IgG Substrate (ready-to-use) Avoid contamination of the ready-to-use substrate solution e. g. by using sterile tips Stopping Solution (ready-to-use) english 12

14 Pos: 27 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/testdurchführ ung/t establauf/ebv: T establ Pos: 28 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/testabl auf/t establ os: 29 /Arbeitsanleitung en ELISA classic/gültig für mehr ere D okumente/t estdurchführung/manuell e T estdurchführung (für ALLE Erreger außer C oxi 7.4 Overview - Test Procedure EN SERION ELISA classic Epstein-Barr Virus EA IgG, VCA IgG/IgM, EBNA1 IgG quantitative In case of IgM detection absorption of rheumatoid factor, see No ; Incubation 15 minutes at room temperature or over night at 4 C sample dilution 1 (patient s samples) Pipette diluted samples and ready-to-use control / standard sera into the microtest wells (100 µl) INCUBATION 60 Min./ 37 C moist chamber WASH (4 x 300 µl DIL WASH )² Pipette conjugate solution APC (100 µl) INCUBATION 30 Min./ 37 C moist chamber WASH (4 x 300 µl DIL WASH )² Pipette substrate solution pnpp (100 µl) INCUBATION 30 Min./ 37 C moist chamber Pipette stopping solution STOP (100 µl) READ EXTINCTION at 405 nm 1 Special dilution buffers for the following SERION ELISA classic tests: Borrelia burgdorferi IgG, IgM, EBV EA IgG, Parvovirus B19 IgM and Hantavirus Puumala IgG, IgM 2 For manual use: tap plate at the end of the wash procedure on paper towel. english 13

15 Pos: 30 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/automatische 7.5 Manual Test Procedure 1. Place the required number of cavities in the frame and prepare a protocol sheet. 2. Add each 100 µl of diluted sample or ready-to-use controls into the appropriate wells of microtiter test strips. Spare one well for substrate blank, e.g.: IgA/IgG/IgM quantitative well no. well A1 well B1 well C1 well D1 Substrate blank Negative Control Standard serum Standard serum well E1 Patient Sample incubation for 60 minutes (+/- 5 min) at 37 C (+/- 1 C) in moist chamber 4. After incubation wash all wells with washing solution (by automated washer or manually): - aspirate or shake out the incubation solution - fill each well with 300 µl washing solution - aspirate or shake out the washing buffer - repeat the washing procedure 3 times (altogether 4 times!) - dry by tapping the microtiter plate on a paper towel 5. Addition of conjugate Add 100 µl of the ready-to-use IgA/IgG/IgM conjugate to the appropriate wells (except substrate blank) 6. Conjugate incubation for 30 minutes (+/- 1 min)* at 37 C (+/- 1 C) in moist chamber. 7. After incubation wash all wells with washing solution (see above) 8. Addition of substrate Add 100 µl of ready-to-use substrate solution to each well (including well for substrate blank!) 9. Substrate incubation for 30 minutes (+/- 1 min)* at 37 C (+/- 1 C) in moist chamber. 10. Stopping of the reaction Add 100 µl stopping solution to each well, shake microtiter plate gently to mix. 11. Read extinction Read optical desity (OD) within 60 minutes at 405 nm against substrate blank, reference wave length between 620 nm and 690 nm (e.g. 650 nm). * Please note, that under special working-conditions internal laboratory adaptations of the incubation times may be necessary. english 14

16 Pos: 31 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/positi vkontroll e / Richtigkeitskontroll Pos: 32 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/kapi tel überschrift: TESTAU SWER TUNG + Erreg 7.6 Automated Test Procedure EN SERION ELISA are suited for processing on automats and evaluated for use with Immunomat TM as well as with DYNEX DSX and DS2. The automated processing is performed analogous to manual use. Please note, that under special working-conditions internal laboratory adaptations of the incubation times may be necessary. 7.7 Positive Control / Accuracy Control For the periodic verification of the test method, in order to fulfil the requirements of laboratory internal quality management systems, we recommend using SERION ELISA controls to determine precision and accuracy of SERION ELISA classic test runs. The use of SERION ELISA controls is described in specific instruction manuals. english 15

17 Pos: 34 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/testauswertung: Testgültig kei 8 TEST EVALUATION SERION ELISA classic EBV VCA IgG/IgM, EBV EBNA1 IgG, EBV EA IgG Pos: 33 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/testauswertung: Ei n-punkt-quantifizi 8.1 Single-Point Quantification with the 4PL Method Optimised assignment of extinction signals to quantitative values is guaranteed by using non-linear functions, which adjust a sigmoide curve without any further transformation to OD-values. Determination of antibody concentrations with the SERION ELISA classic is carried out by the 4 parameter logistic-log-model (4 PL) which is ideal for exact curvefitting. It is based on the formula: OD = A e D - A B(C - ln Conc.) The parameters A, B, C, and D are representative for the exact shape of the curve: 1. lower asymptote parameter A 2. slope of the curve parameter B 3. turning point parameter C 4. upper asymptote parameter D For each lot the standard curve is evaluated by Institut Virion\Serion GmbH (Würzburg, Germany) in repeated test runs under optimal conditions. Time consuming and cost intensive construction of the standard curve by the user is not necessary. For evaluation of antibody concentrations a lot specific standard curve as well as a lot specific evaluation table is included with each SERION ELISA classic test kit. The evaluation software SERION evaluate as well as the Microsoft Excel-based software tool SERION activity are available on request. To compensate for normal test variations and also for test run control a standard serum is used in each individual test run. For this control serum a reference value with a validity range is determined by the quality control of the producer. Within this range a correct quantification of antibody concentration is ensured. english 16

18 Pos: 35 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/auswertung: Ü Pos: 36 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/nichtautomatisierte 8.2 Criteria of Validity EN - The substrate blank must be < 0.25 OD. - The negative control must produce a negative test result. - By use of quantitative SERION ELISA classic tests the mean OD-value (after subtraction of the substrate blank!) of the standard serum must be within the validity range, which is given on the lot specific quality control certificate. - The variation of OD-values of the standard serum may not be higher than 20 %. If these criteria are not met, the test is not valid and must be repeated. 8.3 Calculation SERION ELISA classic EBV VCA IgG/IgM, EBV EBNA1 IgG, EBV EA IgG Non-automated Evaluation For the SERION ELISA classic test evaluation a lot-specific quality control certificate with standard curve and an evaluation table is included in the test kit so that the obtained OD values may be assigned to the corresponding antibody activities. The substrate blank must be substracted from all OD values prior to evaluation. Pos: 37 /Ar bei tsanl eitungen ELISA cl assic/gültig für mehrer e D okumente/t estauswertung/testauswertung: M ethode 1 (für all e T ests auß er T etanus & Di s Method 1: Qualitative Evaluation To fix the cut-off ranges multiply the mean value of the measured standard OD with the numerical data of the quality control certificate (see special case formulas), e.g.: OD = x MW(STD) with upper cut-off OD = x MW(STD) with lower cut-off If the measured mean absorbance value of the standard serum is 0.64 OD, the range of the cut-off is in between OD. english 17

19 Pos: 39 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/automatische Auswer Method 2: Pos: 38 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/testauswertung: Methode 2 (für alle T Continuous Determination of Antibody Activities using the Standard Curve So called interassay variations (day to day deviations and laboratory to laboratory deviations) are compensated by multiplication of the current measured value obtained with a patient s sample with the correction factor F. This factor is calculated as follows: F = OD-reference value (of standard serum) OD-current value (of standard serum) The procedure is necessary to adjust the current test level of the user with the lot-specific standard curve. First, daily deviations have to be corrected by calculating the correction factor F. 1. The mean of the two OD-values of the standard serum has to be calculated and checked that it is within the given validity range. 2. Calculation of the factor F: the given reference value is divided by the mean of the extinction of the standard serum: F = reference value extinction STD serum / mean value extinction STD serum. 3. All measured values of patient s samples are multiplied by F. 4. Antibody activities in IU/ml or U/ml can be determined from the standard curve with the corrected values. english 18

20 Pos: 40 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/quantifi Pos: 41 /Ar bei tsanl eitungen ELISA cl assic/gültig für mehrer e D okumente/t estauswertung/grenzwertber eich ( für T este mehrerer Pos: 42 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/kapi tel überschrift: Interpretation der Automatic Test Evaluation with Software SERION evaluate EN After input of the four parameters and the reference value of the standard serum, antibody activities are calculated online from processed and measured SERION ELISA classic test runs by the evaluation software SERION evaluate. If the optical density of the standard is out of the validity range, the following message will appear. Standard values out of ranges in following groups: Group or Standard values differ more than 20 % in following groups: Group In these cases the test run is invalid and should be repeated. Parameters and reference value need to be changed only if there is a change of lot (evaluation table shows parameters and reference values). Correct input of the lot specific data can be checked on the basis of the standard serum activity (in IU/ml or U/ml) assigned to the standard serum. The calculated mean value of the units has to correspond to the unit value indicated on the lot specific certificate. There is an automatic correction of the measured values. In the standard version the printout displays the following: Sample code OD-value IU/ml or U/ml Evaluation 8.4 Limits of Quantification The limits of quantification are specified on the quality control certificate of the SERION ELISA classic test. The linearity of dilution within this range has been demonstrated in comprehensive evaluation studies. In case a patient sample shows a test result above the upper limit of quantification, the sample may be tested at a higher dilution. The thereby determined antibody activity must be multiplied by the additional dilution factor. 8.5 Borderline Ranges The borderline ranges of the SERION ELISA classic EBV VCA IgG/IgM, EBV EBNA1 IgG, EBV EA IgG tests are specified on the quality control certificates and indicate the range for borderline test results. Values obtained, when testing a patient s sample, which fall below this range indicate a negative test result; values above the borderline range are interpreted positive. In cases where the results are within the borderline range a definitive interpretation of the result is not possible. In such cases, the test should be repeated in parallel with a follow-up sample taken one to two weeks later (serum pair). english 19

21 8.6 Interpretation of Results Pos: 43 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/testauswertung/ebv/ebv: Inter pretation der Diagnosis of primary Infectious Mononucleosis (IM) is mainly by the detection of antibodies to the early antigen (EA) and against the protein complex known as the viral capsid antigen (VCA), the importance of which has been known for some time. Shortly after infection IgM antibodies directed against EA and VCA appear and then, as the acute infection resolves, diminish or disappear completely. In contrast, anti-vca IgG, which is produced at almost the same time, persists life long although the levels of activity may vary. In the case of reactivation (for example following immunosuppression) there is a significant increase in the levels of both anti-ea IgG and anti-vca IgG, and, in rare cases, VCA IgM may be detectable. Patients with Burkitt lymphoma and nasopharyngeal carcinoma have the same characteristic high levels of VCA IgG as seen in reactivations. In addition, the demonstration of VCA IgA antibodies provides supporting evidence in the diagnosis of nasopharyngeal carcinoma. Increased levels of anti-ea antibodies are likewise a characteristic feature in patients suffering from nasopharyngeal carcinoma or experiencing a reactivation. Several weeks following primary infection antibodies directed against the EBV nuclear antigen 1 (EBNA1) appear and remain detectable lifelong although they may disappear if the patient is immunosupressed. Alone, or in combination with VCA IgG antibodies, they are considered a marker for a past infection. A decline in EBNA1 IgG is frequently observed following virus reactivation. This is usually accompanied by an increase in VCA IgG activity while antibodies against early antigen reappear. Borderline results in the SERION ELISA classic EBNA1 IgG test in combination with positive results in the SERION ELISA classic EBV VCA IgG are interpreted as being positive and evidence of a long past infection. In around 25 % of cases of a fresh infection with CMV VCA IgM antibodies are produced which may result in the unusual antibody combination of EBNA1 IgG positive, VCA IgG positive and VCA IgM positive. The presence of EBNA1 IgG however is definitive evidence for a past infection with EBV. In the following table the various possible combinations of antibodies and their interpretation are listed. english 20

22 Pos: 44 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/kapi tel überschrift: Refer enzbereiche gesunder Pr Basic interpretation scheme EN This basic interpretation scheme is mainly for differentiation between a seronegative immune status, primary infection and past infection by using the four most important serological markers EA IgG, VCA IgG, VCA IgM and EBNA1 IgG: EA IgG VCA IgM VCA IgG EBNA1 IgG Interpretation seronegativity acute / recent infection past infection reactivation 8.7 Reference Range of healthy Individuals Pos: 45 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/testauswertung/ebv/ebv: Refer enzbereich gesunder Testing of random blood donor sera, collected in the region of southern Germany, with the SERION ELISA classic EBV VCA IgM / IgG, EBNA1 IgG and EA IgG resulted in the following distribution. From 131 sera tested with the SERION ELISA classic EBV VCA IgM, 131 (100 %) were negative. 126 (96.2 %) were positive when tested with the SERION ELISA classic EBV VCA IgG, three (2.3 %) yielded a positive result and two (1.5 %) were evaluated as borderline. In addition, results from testing 131 sera in the SERION ELISA classic EBNA1 IgG were as follows; 120 sera (91.6 %) tested positive, and 11 (8.4 %) sera were negative. The ELISA classic EA IgG gave the following results: 28 (21.4 %) positive, 11 (8.4 %) borderline and 92 sera (70.2 %) were negative. From these results a seroprevalence in the general population of >96 % is calculated. Pos: 46 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/leistungsmer kmale/kapi tel überschrift Leistungsmer english 21

23 Pos: 47 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/leistungsmer kmale/kapi tel überschrift: Sensiti vität und Spezi 9 PERFORMANCE CHARACTERISTICS 9.1 Sensitivity and Specificity Pos: 48 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/leistungsmer kmal e/ebv/ebv: Sensi ti vität und Spezifi For the evaluation of the SERION ELISA classic EBV VCA IgM, EBV VCA IgG and EBV EBNA1 IgG tests they were tested in a comparison study with several commercially available EBV ELISA tests. SERION ELISA classic EBV EA IgG: The SERION ELISA classic EBV EA IgG was evaluated against four other ELISA tests using a panel of 133 sera of which 38 were positive and 95 negative. As two of the tests (3 & 4) displayed an extremely low sensitivity or specificity, only the other two remaining tests (1 & 2) were used for the comparison study. Discrepant results between these two tests and SERION ELISA classic EBV EA IgG were examined using immunoblot and dependant upon the immunoblot results assigned to the positive or negative serum panel. These serum panels were used to determine sensitivity, specificity, and positive and negative predictive values as well as the efficiency calculation. Borderline results were excluded from the calculation. SERION ELISA classic EBV-EA-IgG Test 1 Test 2 Test 3 Test 4 sensitivity 81.8 % 100 % 75.0 % 30.8 % % specificity 97.6 % 81.0 % 85.7 % 100 % 3.6 % predictive-value (+) 93.1 % 77.6 % 77.4 % 100 % 34.1 % predictive value (-) 93.2 % 100 % 84.0 % 70.0 % 100 % efficiency 93.2 % 88.5 % 81.5 % 73.5 % 35.7 % SERION ELISA classic EBV VCA IgM: 155 serum samples with different antibody reactivities (positive panel of 78 and negative panel of 77 serum samples) were tested in an in house study in comparison to four other commercially available ELISA tests. Sera were defined positive and negative, respectively, when consistent results were found in 4 of 5 assays. Discrepant sera were analyzed by Immunoblot analysis and then categorized negative or positive, dependant upon the Immunoblot results. Sensitivity and specifity were calculated with these positive and negative panels while borderline results were not included in these calculations. english 22

24 Pos: 49 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/leistungsmer kmale/kapi tel überschrift Präzisi SERION ELISA classic EBV VCA IgG: The SERION ELISA classic EBV VCA IgG was evaluated in an external study with 209 positive serum samples and 71 negative serum samples in comparison to immunoblot. Sera classified as borderline were not included into the calculation of sensitivity and specificity. EN SERION ELISA classic EBV EBNA 1 IgG: Evaluation of SERION ELISA classic EBV EBNA 1 IgG was verified in comparison to the test of a competitor using a panel of 130 positive sera and a negative panel of 69 sera. Borderline test results were not included in sensitivity and specificity calculation. The results are summarized in the following table: SERION ELISA classic EBV-VCA IgG assay EBV-VCA IgM assay EBV-EBNA1 IgG assay Sensitivity 98.1 % 97.4 % 98.5 % Specificity 98.5 % 97.3 % > 99 % english 23

25 9.2 Reproducibility Pos: 50 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/leistungsmer kmal e/ebv/ebv: Intraassay reproducibility was determined by testing sera of different reactivities 20 times in one test run. Interassay reproducibility was determined by testing sera of different reactivities in 10 independent test runs. Standard deviation Coefficient of Variation (CV %) = x 100 Mean value SERION ELISA classic EBV EA IgG: Sample Mean Value Intraassay Mean Value Interassay (OD) (CV %) (OD) (CV %) weak positive positive strong positive SERION ELISA classic EBV VCA IgM: Sample Mean Value Intraassay Mean Value Interassay (OD) (CV %) (OD) (CV %) positive strong positive SERION ELISA classic EBV VCA IgG: Sample Mean Value Intraassay Mean Value Interassay (OD) (CV %) (OD) (CV %) weak positive positive strong positive english 24

26 Pos: 51 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/allgemei ne Texte ELISA Pos: 52 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/liter atur/kapitelüberschrift: Liter SERION ELISA classic EBV EBNA1 IgG: EN Sample Mean Value Intraassay Mean Value Interassay (OD) (CV %) (OD) (CV %) weak positive positive strong positive SAFETY MEASURES 10.1 Statements of Warning The SERION ELISA classic is designed for use by qualified personnel who are familiar with good laboratory practice. All kit reagents and human specimens should be handled carefully, using established good laboratory practice. - This kit contains human blood components. Although all control- and cut-off sera have been tested and found negative for anti-hiv-ab, HBs-Ag (Hepatitis B-Virussurface Antigen) and anti-hcv-ab, they should be considered potentially infectious. - Do not pipette by mouth. - Do not smoke, eat or drink in areas in which specimens or kit reagents are handled. - Wear disposable gloves, laboratory coat and safety glasses while handling kit reagents or specimens. Wash hands thoroughly afterwards. - Patient s material and other potentially infectious material should be decontaminated after the test run. - Reagents should be stored safely and be unaccessible to unauthorized access e.g. children. - Stopping solution: corrosive (C); causes acid burn (R34) Use safety glasses, gloves and laboratory coat while handling! 10.2 Disposal Please observe the relevant statutory requirements! english 25

27 Pos: 53 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/literatur /EBV: Li ter ===== Ende der Stückliste ===== 11 REFERENCES [1] Aalto, S. M. (1998) Immunoreactivation of Epstein-Barr Virus Due to Cytomegalovirus Primary Infection. J. Med. Virol. 56, [2] Bauer, G. (1994) Epstein-Barr Virus - Bedeutung und Möglichkeiten der Labordiagnostik. Therapeutische Umschau 51, [3] Bauer, G. (2001) Simplicity Through Complexity: Immunoblot With Recombinant Antigens as the New Gold Standard in Epstein-Barr Virus Serology. Clin. Lab. 47, [4] Linde, A. (1992) Diagnosis and pathogenesis of infectious mononucleosis and other Epstein Barr virus-associated disease. Rev. Med. Microbiol. 3, [5] Porstmann, T. (1992) Epstein-Barr Virus. Diagnostische Bibliothek 6/7, 1-8. [6] Prang, N. S., Schwarzmann, F. (1997) Aktuelle Perspektiven in der Diagnostik Epstein-Barr Virus assoziierter Erkrankungen. Immun. Infekt [7] Seigneurin, J. M. (1992) Diagnostik von Infektionen mit dem Epstein-Barr Virus. Diagnose und Labor 42, english 26

28 Actualizaciones Preste atención a las diferencias en comparación con la versión anterior. Nº de la versión actual: V 22.11/12-1 Versión anterior: V 21.10/01-1 Actualización en la sección: Actualización general, 5, SERION ELISA classic Epstein-Barr Virus VCA IgG/IgM, Epstein-Barr Virus EBNA1 IgG, Epstein-Barr Virus EA IgG CONTENIDO ES 1 USO PREVISTO 2 PERTINENCIA DIAGNÓSTICA 3 PRINCIPIO DE LA PRUEBA SERION ELISA classic 4 COMPONENTES DEL KIT 5 MATERIAL NECESARIO PERO NO SUMINISTRADO 6 CONSERVACIÓN Y ESTABILIDAD 7 PROCEDIMIENTO DE LA PRUEBA SERION ELISA classic 7.1 Evidencia de deterioro 7.2 Preparación y conservación de la muestra 7.3 Preparación de reactivos del kit 7.4 Visión general - procedimiento de la prueba 7.5 Procedimiento manual 7.6 Procedimiento automatizado 7.7 Control positivo / control de exactitud 8 EVALUACIÓN DE LAS PRUEBAS 8.1 Cuantificación de punto simple con el método 4PL 8.2 Criterios de validez 8.3 Cálculos del SERION ELISA classic EBV VCA IgG/IgM, EBV EBNA1 IgG, EBV EA IgG 8.4 Límites de cuantificación 8.5 Intervalos dudosos 8.6 Interpretación de resultados 8.7 Intervalos de referencia de individuos sanos 9 CARACTERÍSTICAS DE FUNCIONAMIENTO 9.1 Sensibilidad y especificidad 9.2 Reproducibilidad 10 MEDIDAS DE SEGURIDAD 10.1 Declaraciones de advertencia 10.2 Eliminación 11 BIBLIOGRAFÍA Nº de la versión actual: V 22.11/12-1 Versión previa: V 21.10/01-1

29 Pos: 1 /Arbeitsanl eitung en ELISA classi c/gültig für nur ein Dokument/U pdate/u pdate: Pos: 3 /Arbeitsanl eitung en ELISA classi c/gültig für nur ein Dokument/Bestell nummern/ebv: Pos: 6 /Arbeitsanl eitung en ELISA classi c/gültig für alle D okumente/elisa cl assic/allgemei ne T exte ELISA cl assic/kapitelüberschrift "Diag nostische 1 Pos: classic/allgemeine 4\mod_ _28.doc 2 /Arbeitsanleitungen Texte classic/einleitung für "Enzymimmunoassay" alle SERION ELISA classic Epstein-Barr Virus VCA IgG/IgM, Epstein-Barr Virus EBNA1 IgG, Epstein-Barr Virus EA IgG Enzimoinmunoensayo para la determinación de anticuerpos humanos Para uso diagnóstico in vitro SERION ELISA classic Epstein-Barr Virus VCA IgG SERION ELISA classic Epstein-Barr Virus VCA IgM Nº de pedido: ESR1361G Nº de pedido: ESR1361M SERION ELISA classic Epstein-Barr Virus EBNA1 IgG Nº de pedido: ESR1362G SERION ELISA classic Epstein-Barr Virus EA IgG Nº de pedido: ESR1363G classic/allgemeine 0\mod_ _28.doc Pos: 4 /Arbeitsanleitungen Texte classic/kapitelüberschrift für alle Dokumente/ELISA 1 USO PREVISTO Pos: 5 /Arbeitsanl eitung en ELISA classi c/gültig für nur ein Dokument/Anwendungsber eich/ebv: Las pruebas de SERION ELISA classic Epstein-Barr Virus EA IgG, VCA IgG, IgM y EBNA1 IgG son inmunoensayos cuantitativos y cualitativos para la detección de anticuerpos humanos en suero o plasma dirigidos frente a componentes del virus Epstein- Barr (VEB). SERION ELISA classic Epstein-Barr Virus EA IgG y SERION ELISA classic EBV VCA IgM se recomiendan para la detección de infecciones agudas o reactivaciones. SERION ELISA classic Epstein-Barr Virus VCA IgG determina infecciones primarias y recientes. La prueba SERION ELISA classic EBNA1 IgG se recomienda para la determinación de infecciones pasadas. 2 PERTINENCIA DIAGNÓSTICA Pos: 7 /Arbeitsanl eitung en ELISA classi c/gültig für nur ein Dokument/Di agnostische Bedeutung/EBV: Diagnostische El virus Epstein - Barr (VEB) es un virus humano patógeno que pertenece a la familia Herpesviridae. Se encuentran en todo el mundo y en común con todos los herpesvirus, la prevalencia en la población general es alta, alcanzando del 90 al 95 % en la población adulta. En los llamados paises en desarrollo, la infección primaria se da generalmente en el primer año de vida y es con frecuencia asintomática. Por contra, en paises con altos niveles de higiene, la infección primaria se da principalmente en adolescentes y adultos jóvenes. El pico de edad de infección se encuentra entre los 15 y 20 años y alrededor del 50 % de los infectados desarrollará una mononucleosis infecciosa. La transmisión se produce primariamente a través de un intercambio de saliva aunque son posibles otras rutas como productos derivados de la sangre o trasplante de médula. Durante la infección primaria las glándulas salivares están implicadas inicialmente y los virus alcanzan la nariz y la garganta vía saliva. En este punto, los síntomas de infección son como los de la gripe. El virus es diseminado a través del cuerpo por los linfocitos B infectados, que están regulados normalmente por el sistema inmune pero cuya proliferación es estimulada por el virus. Estos linfocitos B infectados en sangre periférica son característicamente atípicos, con una forma variable, citoplasma claramente basófilo y un núcleo evidente. A medida que la enfermedad avanza puede presentar fiebre alta, esplenomegalia, linfadenitis, trombocitopenia y hepatitis. Debido a la diseminación del virus y la estrategia de transmisión, primeramente vía saliva, la mononucleosis infecciosa (MI) o fiebre glandular se ha dado en llamar también la enfermedad del beso. español 2

30 En casos raros una infección aguda de MI puede conducir a un estado de enfermedad activa crónica. En dichos casos los síntomas de MI pueden continuar durante un período de tiempo considerable. La patogénesis de esta complicación no está clara aunque se sospecha una predisposición genética y/o infección con una cadena particularmente lítica de virus. ES Otro estado raro de enfermedad es la MI crónica que, en contraste con una infección aguda crónica, después de la resolución de los síntomas y de un período de latencia, que dura posiblemente varios años, se reactiva con consecuencias fatales en algunas ocasiones. Las razones y las causas tras esos casos no están claras. Algunas enfermedades genéticas tales como el síndrome linfático proliferativo ligado al cromosoma X, (XLPS, x-linked lymphoproliferative syndrome) pueden dar como resultado una incontrolada proliferación de linfocitos B después de la infección por VEB y frecuentemente conduce a una infección crónica. La supresión médica del sistema immune como la utilizada en pacientes trasplantados puede conducir a la reactivación de virus latentes o a un incremento de la posibilidad de una infección primaria que puede conducir a un rechazo del trasplante inducido por VEB. Los pacientes con SIDA son también, como consecuencia de su enfermedad immune subyacente, más susceptibles a la infección y las reactivaciones. En regiones geográficas concretas, el VEB está estrechamente asociado con la prevalencia de carcinoma nasofaríngeo. Este carcinoma de la faringe, nariz y garganta consta de células epiteliales indiferenciadas y tiene propensíon a la metástasis. Esta enfermedad se presenta globalmente pero es particularmente común en ciertas regiones del sur de China. La predisposición genética así como los aspectos ambientales como la dieta están bajo discusión como cofactores. El linfoma de Burkitt (LB), es también un tumor asociado a VEB, primariamente en las regiones geográficas de África y Papúa Nueva Guinea. Este tumor de linfocitos B monoclonales está también ligado a las zonas con alta prevalencia de malaria y por encima del 90 % de dichos tumores presentan evidencia de virus E-B. La influencia de la infección por plasmodium en la respuesta inmune trae a discusión el papel de la malaria como un cofactor de LB. Se dan casos esporádicos de LB en otras regiones, de cualquier forma en dichos casos, el VEB es mucho menos frecuentemente detectado y se piensa que otros cofactores como los cambios genéticos a través de translocación cromosómica son responsables del tumor. La respuesta de anticuerpos a la infección por VEB es extremadamente variable como consecuencia de la complejidad de los virus y lo heterogéneo de las distintas etapas de la infección. Durante la fase activa de infección, se producen anticuerpos dirigidos a aproximadamente 100 diferentes antígenos virales. Esto decae hasta cerca de 10 durante la infección latente (EBNA 1-6, proteínas de membrana tardías 1-3). Con estos antecedentes ha habido una tendencia hacia el desarrollo de sistemas de detección de anticuerpos que utilizan componentes simples / proteínas más que el virus completo. De esta manera es possible correlacionar la producción de anticuerpos frente a antígenos concretos con las diferentes fases de enfermedad y la progresión de la enfermedad. español 3

31 Symptoms Heterophile ab VCA-IgM weeks months years Figura 1: Títulos de anticuerpos tras la infección por VEB. (Fuente: La Figura 1 muestra la típica respuesta inmune en terminos de producción de anticuerpos tras la infección por VEB. IgM se produce muy temprano en la infección contra el antígeno temprano (EA). Las concentraciones máximas de anticuerpos tienden a coincidir con la aparición de los síntomas, y aproximadamente dos semanas después de la infección inicial comienza la producción de IgG para EA, IgM para VCA (Virus Capsid Antigen) e IgG para VCA. Las concentraciones más altas de IgM anti-vca, se encuentran en torno a tres semanas después de la aparición de síntomas. Las IgG frente a EA son frecuentemente detectables durante un tiempo considerable tras la infección. En consecuencia, las concentraciones de IgM frente a VCA y más tarde de IgG frente a EA decaen. La IgG anti-vca alcanza un pico unas seis semanas tras la aparición de los síntomas y permanece en un nivel alto durante toda la vida. Alrededor de tres semanas después de la aparición de los síntomas los anticuerpos IgG frente a EBNA1, que son un indicador de una infección pasada, empiezan a producirse y alcanzan un pico en torno al mes siete y permanencen en un nivel alto tras una infección normal por VEB. La reactivación de los virus (como la immunosupresión) normalmente conduce a un significativo incremento de anticuerpos IgG frente a EA mientras que los títulos de IgM frente a VCA aumentan. Sólo muy ocasionalmente, después de la inmunosupresión, IgG frente a EBNA1 desaparece de forma que IgG frente a EA es un buen marcador de reactivación. Pos: 8 /Arbeitsanl eitung en ELISA classi c/gültig für alle D okumente/elisa cl assic/t estprinzip/testpri nzip ELISA 1 español 4