SERION ELISA classic Tetanus IgG

Transcript

1 YOUR GLOBAL PARTNER IN DIAGNOSTICS 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 Tetanus IgG SERION ELISA classic Tetanus IgG Instructions - English Instrucciones de empleo - Español Oδηγίες χρήσης - Ελληνικά Instruções de emprego - Português Pokyny - Česky (Version/Versión/Έκδοση/Versão/Verze 14.10/01-1) KAL108-2

2 Updates Please pay attention to the differences in comparison to the previous version. Current version Nr.: V 14.10/01-1 Previous version: V 13.04/12-1 Update in section: General Update SERION ELISA classic Tetanus IgG CONTENTS 1 INTENDED USE EN GR ES 2 DIAGNOSTIC RELEVANCE 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 CZ PT 7.1 Evidence of Deterioration 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 Tetanus IgG 8.4 Limits of Quantification 8.5 Interpretation of Results 8.6 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 14.10/01-1 previous version: V 13.04/12-1

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: 7 /Arbeitsanl eitung en ELISA classi c/gültig für nur ein Dokument/Di agnostische Bedeutung/T etanus: Di agnostische Pos: 8 /Arbeitsanl eitung en ELISA classi c/gültig für alle D okumente/elisa cl assic/t estprinzip/testpri nzip ELISA Pos: 4\mod_ _18.doc 2 /Arbeitsanleitungen Texte classic/einleitung classic/gültig für "Enzymimmunoassay" alle Dokumente/ELISA SERION ELISA classic Tetanus IgG Enzyme-immunoassay for determination of human antibodies for in vitro diagnostic use Dokument/Bestellnummern/Tetanus: 0\mod_ _18.doc Pos: 3 /Arbeitsanleitungen 9164 classic/gültig für ein SERION ELISA classic Tetanus IgG Order Nr.: ESR108G 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/t etanus: SERION ELISA classic Tetanus IgG test is a quantitative immunoassay for the detection of human antibodies in serum or plasma directed against the tetanus toxin. The SERION ELISA classic Tetanus IgG is recommended for determination of the anti-tetanus toxin titer, for vaccination control and for the determination of the current individual immune status to prevent hyperimmune reactions. 2 DIAGNOSTIC RELEVANCE Clostridium tetani, the causative agent of tetanus, is an ubiquitous obligate anaerobic bacterium, that may live in the intestines of many animals and, occasionally, humans without causing disease or inducing immunity. The extremely resistant spores reach the ground through faeces and are able to survive here for years. The pathogen enters the host by way of contaminated wounds. In the presence of anaerobic conditions spores will develop into the vegetative form which produces tetanospasmin, a highly potent neurotoxin, which produces intense muscle spasms. After an incubation period ranging from a few days up to several weeks, initial symptoms are nonspecific symptoms (fatigue, headache, dizziness, muscle pain etc.). This is followed by characteristic cramps which spread craniofacial to caudal and may also affect the respiratory muscles. Sufferers are fully conscious during bouts of these extremely painful cramps which may be triggered by the slightest external stimuli. During the course of the disease the patient may suffer from renal, cardiac and circulatory failure, and disseminated intravasal coagulation (DIC). Even with intensive care, over one third of patients die. The most effective prophylaxis against tetanus is active immunization with an effective tetanus toxoid vaccine which provides good protection against the disease. Side effects of immunization can occur after multiple vaccinations (repeated immunization or booster shots), varying from local and systemic allergic reactions to anaphylactic shock. To check for immune status, serological detection of antitoxin IgG antibodies in the form of ELISA techniques are used. Testing is indicated in cases of; uncertain immune status, possibly due to poor recollection or incomplete documentation, vaccination complications and immune suppression. In testing for immunity the current level of anti-toxin antibodies are determined in Interrnational Units so allowing for informed advice on immunization to be given. english 2

4 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. Pos: 9 /Arbeitsanleitungen ELISA classic/gültig für mehrere Dokumente/Inhalt und Zusammensetzung/Inhalt und Zusammensetzung (alle außer Bor,EBV,Hanta,Parvo, english 3

5 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. 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. 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. 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. Dilution buffer DILB, Protein containing phosphate buffer with Tween 20; preservative: < 0.1 % sodium azide; colouring: 0.01 g/l Bromphenol blue. Stopping solution STOP, 1.2 N sodium hydroxide. 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!) Quality control certificate with standard curve and evaluation table INFO, (quantification of antibodies in IU/ml or U/ml). 2 x 2 ml 2 ml 13 ml 33,3 ml 2 x 50 ml 15 ml 13 ml 2 pages Pos: 10 /Arbeitsanleitungen ELISA classic/gültig für mehrere Dokumente/Zusätzlich benötigte Materialien/Zusätzlich benötigte Materialien (für Teste ohne english 4

6 5 MATERIAL REQUIRED BUT NOT SUPPLIED EN - common laboratory equipment - photometer for microtiter 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 5

7 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 6

8 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 7

9 Pos: 15 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/kapi tel überschrift Pos: 18 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/pr obenlager 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: Pos: 16 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/testdurchführ ung/probenverdünnung/tetanus: V 1 + V 2 = add 10 µl patient s sample each to 1000 µl dilution buffer At OD values over the measurement accuracy decreases as the optical density increases. Consequently, in cases where the OD value is above 2.000, we advise that a further serum dilution of should be tested. This ensures that an accurate determination of the IU/ml value can be made even in sera with high antibody levels. When such patient s samples do present, an initial dilution of can be prepared and tested. For the accurate determination antibody levels in the lower range, all sera with < 1 IU/ml can be tested again at a dilution of V 1 + V 2 = add 10 µl patient s sample each to 1000 µl dilution buffer (= ) 20 µl of first dilution each to 180 µl dilution buffer (= 1 + 9) 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 8

10 Pos: 20 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/reagenzienvorberei tung, T eil Pos: 21 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/testabl auf/ü berschrift Sample Storage EN 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: 19 /Arbeitsanleitungen ELISA classic/gültig für alle Dokumente/ELISA classic/testdurchführung/reagenzienvorbereitung, Teil 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 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) 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 9

11 Pos: 22 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/testdurchführ ung/t establauf/tetanus: T Pos: 23 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/testabl auf/t establ Pos: 24 /Ar bei tsanl eitungen ELISA cl assic/gültig für mehrer e D okumente/t estdurchführ ung/m anuelle Testdurchführ ung (für ALLE Erreg er auß er C 7.4 Overview - Test Procedure SERION ELISA classic Tetanus IgG quantitative sample dilution 1 (patient s samples) bzw 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 10

12 Pos: 25 /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.: EN IgG 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 IgG 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 11

13 Pos: 26 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/positi vkontroll e / Richtigkeitskontroll Pos: 27 /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 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 12

14 Pos: 29 /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 Tetanus IgG Pos: 28 /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 EN 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 13

15 Pos: 30 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/auswertung: Ü Pos: 32 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/nichtautomatisierte 8.2 Criteria of Validity - 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 Tetanus IgG Pos: 31 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/testauswertung/t etanus/tetanus: The indication of the IgG antibody activity in IU/ml refers to the 1 st International Standard for Tetanus immunoglobulin, human (Code: TE-3). The preparation of the standard serum is declared with an activity of 120 IU/vial. For the sensitivity range of 0.05 to 5.0 IU/ml antitoxin and a serum dilution of , a standard curve and an evaluation table are enclosed. The obtained OD values may be assigned to the corresponding antibody activity due to different techniques. If sera have been diluted for the test run, the obtained IU/ml values have to be multiplied by factor 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: 33 /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) english 14

16 Pos: 34 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/automatische Auswer Pos: 35 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/quantifi 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. EN 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 Automatic Test Evaluation with Software SERION evaluate 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 english 15

17 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. Pos: 36 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/kapi tel überschrift: Interpretation der 8.5 Interpretation of Results Pos: 37 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/testauswertung/t etanus/tetanus: Inter pretati on der The following table contains the recommendations for interpretation of vaccination titers according to Prof. Dr. med. W. D. Kuhlmann, Ernst-Rodenwald-Insitute, Koblenz, Germany: Value: evaluation of vaccination titers <0.01 IU/ml - no immunity - depending on vaccination anamnesis: booster shot or basic immunization IU/ml - immune protection is not ensured - booster shot recommended; - serologic control after 4-6 weeks IU/ml - adequate immune protection - control of antitoxin level after about 2 years recommended - booster shot leads to long term immune protection IU/ml - adequate immune protection - control of antitoxin level after about 2 years recommended - booster shot not necessary - Note: if antitoxin titer > 0,5 IU/ml, vaccination can cause side effects IU/ml - long term protection - titer control after 5-10 years > 5.0 IU/ml - long term protection - titer control after about 10 years english 16

18 Pos: 38 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estauswertung/kapi tel überschrift: Refer enzbereiche gesunder Pr Pos: 40 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/leistungsmer kmale/kapi tel überschrift Leistungsmer Pos: 41 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/leistungsmer kmale/kapi tel überschrift: Sensiti vität und Spezi Pos: 43 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/leistungsmer kmale/kapi tel überschrift Präzisi Please note that values in international units listed above were obtained in an in vivo mouse-test for the detection of antibodies against tetanus toxin. EN Values below 0.1 U/ml, obtained with the ELISA methodology, are below the level of technically reproducible sensitivity. The vaccination committee STIKO of the Robert-Koch-Institute, Berlin, Germany published the following in July 2001 recommending vaccination for: All persons with no or insufficient basic immunization or those who received a booster shot more than ten years ago. For further information, such as tetanus vaccination following injury, please refer to the current vaccination recommendations of STIKO at Robert-Koch-Institute, Berlin, Germany 8.6 Reference Range of healthy Individuals Pos: 39 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/testauswertung/t etanus/tetanus: R eferenzber eich gesunder Pr Testing of random blood donor sera, collected in the region of southern Germany, with the SERION ELISA classic Tetanus IgG test resulted in the following distribution. From 88 sera tested 82 had anti-tetanus toxoid antibody levels over 0.1 IU/ml so indicating that 93.2 % of the blood donors tested showed an effective immunity to tetanus. This result is in agreement with the published literature which reports that in Germany 96 % of men and 71 % of women have an antibody level of >0.1 IU/ml. These figures also include older individuals between the ages of 60 and 70 years who were not included in the blood donor panels and evidently retain immunity many years after immunization. 9 PERFORMANCE CHARACTERISTICS 9.1 Sensitivity and Specificity Pos: 42 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/leistungsmer kmal e/t etanus/tetanus: Sensi ti vität und Results of the determination of immune status are expressed in IU/ml and the level of antibody allows for an informed estimate of the expected duration of immune protection. Sensitivity, specificity and predictive values cannot be calculated as the aim of the test is not the discrimination between positive and negative results but a continuous quantitative determination of antibody activities. This kind of test evaluation is common for vaccine control ELISA systems. The Central Institute of Medical Services of the German Federal Armed Forces in Koblenz (medical-immunology, Prof. Dr. med. Kuhlmann) performed the test evaluation for SERION ELISA classic Tetanus IgG. An internal ELISA test from this institute was also performed as a comparison. The results of testing 269 sera with the two tests demonstrated a high level of agreement between the two across the complete measurement range. A correlation of 0.94 was found in a range of IU/ml. english 17

19 Pos: 45 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/allgemei ne Texte ELISA 9.2 Reproducibility Pos: 44 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/leistungsmer kmal e/t etanus/tetanus: Präzi 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 assays performed on 5 different days. Standard deviation Coefficient of Variation (CV %) = x 100 Mean value SERION ELISA classic Tetanus IgG Sample Mean Value Intraassay Mean Value Interassay (OD) (CV %) (OD) (CV %) weak positive positive strong positive english 18

20 Pos: 46 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/liter atur/kapitelüberschrift: Liter 10 SAFETY MEASURES EN 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 19

21 Pos: 47 /Ar bei tsanl eitungen ELISA cl assic/gültig für nur ein D okument/literatur /Tetanus: ===== Ende der Stückliste ===== 11 REFERENCES [1] Kuhlmann W. D. (1991) Tetanus. Impfung, Impftiter und Impfreaktion. Zentrales Institut des Sanitätsdienstes der Bundeswehr Koblenz. [2] Müller H.E., Müller M., Schick W. (1988) Tetanus-Schutzimpfung-Indikation und Kontraindikation. Dtsch. med. Wschr. 113, [3] Pietsch M. (1991) Indikationsbeispiele für die Tetanus-Antikörper-Bestimmung. Fortschritte der Diagnostik 1, [4] Pietsch M., Geißler E., Pietsch R., Borneff J. Tetanuserkrankung-Klinik und Impfprophylaxe. Ärzteblatt Rheinland-Pfalz 41, [5] Pietsch M., Pietsch R. (1989) Einsatz der ELISA-Technik zur Messung antitoxischer Tetanus-Antikörper. Wehrmed. Mschr. 6, [6] Robert-Koch-Institut (2001) Impfempfehlungen der Ständigen Impfkommission (STIKO) am Robert Koch-Institut. Epidemiologisches Bulletin 28. [7] Schröder J.P., Kuhlmann W.D. (1991) Tetanusimmunität bei Männern und Frauen in der Bundesrepublik Deutschland. Immun. Infekt. 19, [8] Schröder J. P., Kuhlmann W. D. (1992) Tetanusimpfschutz und Vermeidung von Nebenreaktionen bei Auffrischimpfungen. Dtsch. med. Wschr. 117, [9] Staak M., Wirth E. (1973) Zur Problematik anaphylaktischer Reaktionen nach aktiver Tetanusimmunisierung. Dtsch. med. Wschr. 98, english 20

22 Actualizaciones Preste atención a las diferencias en comparación con la versión anterior. Nº de la versión actual: V 14.10/01-1 Versión anterior: V 13.04/12-1 Actualización en la sección: Actualización general SERION ELISA classic Tetanus 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 Tetanus IgG 8.4 Límites de cuantificación 8.5 Interpretación de resultados 8.6 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 14.10/01-1 Versión previa: V 13.04/12-1

23 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 1 Pos: classic/allgemeine 4\mod_ _28.doc 2 /Arbeitsanleitungen Texte classic/einleitung für "Enzymimmunoassay" alle SERION ELISA classic Tetanus IgG Enzimoinmunoensayo para la determinación de anticuerpos humanos Para uso diagnóstico in vitro Pos: 3 /Arbeitsanl eitung en ELISA classi c/gültig für nur ein Dokument/Bestell nummern/tetanus: SERION ELISA classic Tetanus IgG Nº de pedido: ESR108G Pos: classic/allgemeine 0\mod_ _28.doc 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/t etanus: Las pruebas de SERION ELISA classic Tetanus IgG son inmunoensayos cuantitativos para la detección de anticuerpos humanos en suero o plasma dirigidos frente a la toxina tetánica. El SERION ELISA classic Tetanus IgG está recomendado para la determinación del título de la toxina antitetánica, para el control de la vacunación y para la determinación del estado inmunológico individual actual y para prevenir reacciones de hiperinmunidad. 2 PERTINENCIA DIAGNÓSTICA Pos: 7 /Arbeitsanl eitung en ELISA classi c/gültig für nur ein Dokument/Di agnostische Bedeutung/T etanus: Di agnostische Clostridium tetani, el agente causante del tétanos, es una bacteria anaerobia obligada ubicua, que puede vivir en el intestino de muchos animales y, ocasionalmente, de los seres humanos sin producir una enfermedad ni inducir inmunidad. Las esporas, extremadamente resistentes, alcanzan el suelo a través de las heces y son capaces de sobrevivir allí durante años. El patógeno se introduce en el hospedador por heridas contaminadas. En presencia de condiciones anaeróbicas, las esporas se desarrollarán en su forma vegetativa, la cual produce tetanospasmina, una neurotoxina muy potente, que produce espasmos musculares intensos. Después de un período de incubación que varía desde algunos días hasta varias semanas, los síntomas iniciales son síntomas no específicos (fatiga, cefalea, mareo, dolor muscular etc.). A esto le siguen característicos calambres que avanzan en sentido craneofacial a caudal y también pueden afectar a los músculos implicados en la respiración. Quienes la padecen son totalmente conscientes durante los ataques de estos calambres extremadamente dolorosos que pueden ser disparados por el más mínimo estímulo externo. Durante el transcurso de la enfermedad, el paciente puede sufrir deterioro renal, cardiaco y circulatorio, y coagulación intravasal diseminada (CID). Incluso con cuidados intensivos, más de un tercio de los pacientes mueren. La profilaxis más eficaz frente al tétanos es la inmunización activa con una eficaz vacuna toxoide tetánica que proporciona una buena protección frente a la enfermedad. Los efectos adversos de la inmunización pueden ocurrir tras varias vacunaciones (Inmunización repetida o revacunaciones), que varían desde reacciones alérgicas locales y sistémicas hasta choque anafiláctico. Para comprobar el estado inmunológico, se utiliza la detección serológica de anticuerpos IgG para la antitoxina en forma de técnicas de ELISA. Las pruebas están indicadas en casos de; estado inmunológico incierto, posiblemente debido a un historial deficiente o una documentación incompleta, complicaciones de la vacunación y supresión inmune. En las pruebas de la inmunidad el nivel actual de anticuerpos anti-toxina se determina en Unidades Internacionales de modo que se puede proporcionar un asesoramiento informado sobre la inmunización. Pos: 8 /Arbeitsanl eitung en ELISA classi c/gültig für alle D okumente/elisa cl assic/t estprinzip/testpri nzip ELISA 1 español 2

24 3 PRINCIPIO DE LA PRUEBA SERION ELISA classic La prueba ELISA (Enzyme Linked Immunosorbent Assay, análisis de inmunoabsorción ligado a enzimas) es un inmunoensayo, que es particularmente apropiado para la determinación de anticuerpos en el campo de la serología infecciosa. La reacción se basa en la interacción específica de anticuerpos con su antígeno correspondiente. Las tiras reactivas de la placa de microtitulación de SERION ELISA classic se recubren con antígenos específicos del agente patógeno de interés. Si los anticuerpos en la muestra de suero del paciente están presentes, se unen al antígeno fijado. Un anticuerpo secundario, que se ha conjugado con la enzima fosfatasa alcalina, detecta y se une al complejo inmune. El sustrato incoloro p-nitrofenolfosfato se convierte entonces en el producto coloreado p-nitrofenol. La intensidad de la señal de este producto de reacción es proporcional a la concentración del analito en la muestra y se mide fotométricamente. Pos: 9 /Arbeitsanleitungen ELISA classic/gültig für mehrere Dokumente/Inhalt und Zusammensetzung/Inhalt und Zusammensetzung (alle außer Bor,EBV,Hanta,Parvo, 1 ES español 3

25 4 COMPONENTES DEL KIT Componentes de la prueba partes / Volumen Pocillos de microtitulación en tiras separables, cada una de ellas con ocho pocillos individuales recubiertos de antígeno (En total son 96) MTP, 1 bastidor. El material de recubrimiento está inactivado. 12 partes Suero patrón (listo para usar) STD, Suero humano en tampón fosfato que contiene proteína; negativo para Ac anti-vih, Acs HB (HBs-Ag, antígeno de superficie del virus de la hepatitis B) y Ac anti-vhc; conservante: azida sódica < 0,1 %; colorante: Amaranto O. 2 x 2 ml Suero control negativo (listo para usar) NEG, Suero humano en tampón fosfato que contiene proteína; negativo para Ac anti-vih, Acs HB (HBs-Ag, antígeno de superficie del virus de la hepatitis B) y Ac anti-vhc; conservante: azida sódica < 0,1 %; colorante: Verde Lissamin Green V. 2 ml Conjugado FA de IgA, IgG o IgM anti-humana (listo para usar) APC, Anticuerpo policlonal IgA, IgG o IgM anti-humano, conjugado con fosfatasa alcalina, estabilizado con solución que contiene proteínas; conservante: metilisotiazolona al 0,01 %, bromonitrodioxano al 0,01 %. 13 ml Concentrado de dilución de lavado (suficiente para 1000 ml) WASH, Solución de cloruro de sodio con Tween 20 y 30 mm Tris/HCl, ph 7,4; conservante: azida sódica < 0,1 %. 33,3 ml Solución amortiguadora de dilución DILB Tampón fosfato con Tween 20 que contiene proteína; conservante: azida sódica < 0,1 %; colorante: 0,01 g/l de azul de bromofenol. 2 x 50 ml Solución de parada STOP, hidróxido de sodio 1,2 N. 15 ml Sustrato (listo para usar) pnpp, Para-nitrofenilfosfato en solución amortiguadora sin disolvente; conservante: azida sódica < 0,1 % ( El sustrato en un frasco sin abrir puede tener una coloración ligeramente amarilla, lo que no reduce la calidad del producto!) 13 ml Certificado de control de calidad con curva patrón y tabla de evaluación INFO, 2 páginas (cuantificación de anticuerpos enui/ml o U/ml). Pos: Materialien/Zusätzlich 5\mod_ _28.doc 10 /Arbeitsanleitungen benötigte Materialien (für Teste für mehrere ohne IgM-Nachweis) benötigte español 4

26 5 MATERIAL NECESARIO PERO NO SUMINISTRADO - equipo habitual de laboratorio - fotómetro para placas de microtitulación con filtro, longitud de onda 405 nm, longitud de onda de referencia recomendada 620 nm nm (p.ej., 650 nm) - incubador a 37 C - cámara de humectación - agua destilada - Clips de cierre por presión (Click-Clips, nº de pedido VT120) Pos: 11 /Arbeitsanleitungen ELISA classic/gültig für alle Dokumente/ELISA classic/lagerung und Haltbarkeit/Lagerung und 1 es ES español 5

27 Pos: 12 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/überschrift: Durchführ 1 6 CONSERVACIÓN Y ESTABILIDAD Reactivo Conservación Estabilidad Tiras de microtitulación (recubiertas con antígeno) sin abrir tras la apertura entre 2 8 C en una bolsa de aluminio cerrada con desecante ver fecha de caducidad; período de conservación mínimo: cuatro semanas; Sueros control / Sueros patrón Las tiras que no se utilizan se deben conservar secas en la bolsa de aluminio cerrada. tras la apertura entre 2 8 C período de conservación en caso de utilización y conservación adecuadas hasta la fecha de caducidad ver fecha de caducidad; 24 meses desde su producción Conjugado solución lista para usar entre 2 8 C Evite la contaminación p.ej., utilizando puntas estériles. ver fecha de caducidad; 28 meses desde su producción Solución amortiguadora de dilución Sin abrir tras la apertura entre 2 8 C Deseche las soluciones turbias. ver fecha de caducidad; 36 meses desde su producción; 24 meses Solución de lavado Concentrado tras la apertura entre 2 8 C Sustrato dilución de trabajo entre 2 8 C dilución de trabajo a temperatura ambiente Los frascos utilizados para la dilución de trabajo se deben limpiar regularmente. Deseche las soluciones turbias. solución lista para usar entre 2 8 C, conservar protegida de la luz Evite la contaminación p.ej., utilizando puntas estériles. Desechar si la solución se vuelve amarilla (extinción frente al agua destilada > 0,25 DO). ver fecha de caducidad; 2 semanas; 1 semana ver fecha de caducidad; 36 meses desde su producción Solución de parada Tras la apertura a temperatura ambiente ver fecha de caducidad español 6

28 Pos: 13 /Ar bei tsanl eitungen ELISA cl assic/gültig für all e D okumente/elisa classic/t estdurchführung/allgemeine Hi nweise ELISA cl 2 7 PROCEDIMIENTO DE LA PRUEBA SERION ELISA classic 7.1 Evidencia de deterioro Utilice únicamente reactivos SERION ELISA classic al utilizar inmunoensayos SERION ELISA classic. Los componentes no deben intercambiarse con reactivos de otros fabricantes. Los sueros patrón y control de los inmunoensayos SERION ELISA classic immunoassays están definidos exclusivamente para el kit de pruebas que se va a utilizar y no deben utilizarse en otros lotes. La solución amortiguadora de dilución, la solución de lavado, el sustrato y la solución de parada se pueden utilizar para todos los inmunoensayos SERION ELISA classic Independientemente del lote y de la prueba. ES Existen tres concentraciones de conjugado diferentes para cada clase de inmunoglobulina: BAJO, MEDIO, ALTO. La clasificación aparece escrita en cada etiqueta como sigue: p.ej. IgG + IgG ++ IgG +++ Conjugado de IgG de baja concentración Conjugado de IgG de media concentración Conjugado de IgG de alta concentración En raros casos es necesaria la utilización de un conjugado especial para garantizar una calidad constante de nuestros productos. Los conjugados especiales se producen en un lote por separado y no llevan el signo + y no son intercambiables con otros conjugados. Preste mucha atención a la información de las etiquetas! Si no han sido abiertos, todos los componentes de las pruebas SERION ELISA classic pueden, si se conservan como corresponde, ser utilizados hasta las fechas de caducidad proporcionadas en las etiquetas. Los reactivos no se pueden utilizar después de la fecha de caducidad. La dilución o alteración de los reactivos puede dar como resultado una pérdida de sensibilidad. Evite la exposición de los reactivos a la luz intensa durante la conservación e incubación. Los reactivos deben estar firmemente cerrados tras su uso para evitar evaporación y contaminación. Para abrir la bolsa de aluminio de la placa de microtitulación, corte la parte superior del lado marcado únicamente, para garantizar poder cerrarla de nuevo adecuadamente. No utilice las tiras si la bolsa de aluminio está dañada o si la bolsa con las tiras restantes y el desecante no ha sido cerrada de nuevo adecuadamente. Utilice técnicas asépticas al retirar alícuotas de los tubos de reactivos para evitar la contaminación. Para evitar resultados falsos positivos, asegúrese de no entrar en contacto o salpicar las paredes superiores de los pocillos mientras se pipetea el conjugado. Tenga cuidado para no mezclar los tapones de los frascos y/o viales. español 7