SERION ELISA antigen Candida

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 antigen Candida SERION ELISA antigen Candida Instructions - English Instrucciones de empleo - Español Oδηγίες χρήσης - Ελληνικά Instruções de emprego - Português Pokyny - Česky (Version/Versión/Έκδοση/Versão/Verze 1.12/09-1) KAL200-2

2 SERION ELISA antigen Candida EN CONTENTS 1 INTENDED USE 2 DIAGNOSTIC RELEVANCE 3 SERION ELISA antigen - TEST PRINCIPLE 4 KIT COMPONENTS GR ES 5 MATERIAL REQUIRED BUT NOT SUPPLIED 6 STORAGE AND STABILITY 7 TEST PROCEDURE SERION ELISA antigen 7.1 General Notes 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 CZ PT 8 TEST EVALUATION 8.1 Single-Point Quantification with the 4PL Method 8.2 Criteria of Validity 8.3 Calculation SERION ELISA antigen Candida 8.4 Limits of Quantification 8.5 Borderline Range 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 1.12/09-1 previous version: --

3 Pos: 1 /Arbeitsanl eitung en ELISA antigen/gültig für alle Dokumente/ELISA antigen/allgemei ne Texte ELISA antigen/einl eitung " Enzymimmunoassay" C andi da Pos: 5 /Arbeitsanl eitung en ELISA antigen/gültig für alle Dokumente/ELISA antigen/allgemei ne Texte ELISA antigen/kapitelüberschrift "Diag nostische 1 SERION ELISA antigen Candida Enzyme-immunoassay for determination of Candida Antigen for in vitro diagnostic use Pos: 2 /Arbeitsanl eitung en ELISA antigen/gültig für nur ei n D okument/bestellnummer n/c andida antigen: Bestell SERION ELISA antigen Candida Order Nr.: ESR200 Pos: antigen/allgemeine 8\mod_ _18.doc 3 /Arbeitsanleitungen Texte antigen/kapitelüberschrift 1 für alle Dokumente/ELISA 1 INTENDED USE Pos: 4 /Arbeitsanl eitung en ELISA antigen/gültig für nur ei n D okument/anwendungsbereich/c andi da antigen: SERION ELISA antigen Candida is a quantitative and qualitative immunoassay for the detection of Candida antigen in human serum or plasma. The assay is recommended for the detection of systemic candidosis. 2 DIAGNOSTIC RELEVANCE Pos: 6 /Arbeitsanl eitung en ELISA antigen/gültig für nur ei n D okument/di agnostische Bedeutung/Candida antigen: Diag nostische albicans is an ubiquitous yeast which, like all Candida species, belongs to the family of yeast-like fungi. Apart from the yeast form which primarily causes superficial infections, so called pseudo mycelia are a further morphologic manifestation of yeast-like fungi. Germ tubes and the development of pseudo mycelia mainly occur in cases of systemic mycosis. Candida ssp. produce and excrete a range of destructive enzymes that enable the facultative pathogen microorganisms to penetrate mucous membrane barriers and blood vessels barriers. Candida ssp. are primarily transmitted by smear contamination from person to person. The primary portal of entry is the oral cavity. Changes in the fungistatic properties of the skin, which are a consequence of a slightly acidic ph value and the antagonistic bacterial flora, can facilitate the establishment of superficial candidiasis of the skin surface. Systemic mycosis results from colonization of mucous membranes, particularly in the gastrointestinal tract. Candida ssp. are able to adhere to the epithelia of a variety of mucosal membranes by adherence proteins and other cell surface structures. The schematic below shows hypothetical steps that may lead to disseminated infections in cases of severe underlying conditions. An exact description of the various stages is not practical (Fig. 1). english 2

4 colonization of the nasopharyngeal tract passing of the stomach lumen transfer to the intestine lumen EN entry into blood circulation transfer to lymphoid system persorption of Candida - ssp. invasion of multiple organs Figure 1: progression of Candida-infections Candidiasis can generally be classified into two major groups whose main characteristics are listed in the table below. superficial Candida-infections deep mycosis (invasive) localization skin, mucosa prime organs immunocompetence of the host risk factors clinical progression + +,-/- pregnancy diabetes AIDS dysfunction of the skin deficient oral hygiene harmless in most cases, no danger of life chemo therapy iatrogene immune suppression central vein catheter radiotherapy high-grade burns surgery life-threatening in most cases therapy effective therapy effectiveness of therapy depends on stage of infection Table 1: Characteristics of Candida-infections english 3

5 Pos: 8 /Arbeitsanl eitung en ELISA antigen/gültig für nur ei n D okument/inhalt und Z usammensetzung/c andi da antigen: Inhalt und 1 The diagnosis of candidiasis on the basis of serological methods is not straight forward: On the one hand transient yeast colonization may induce an antibody response, on the other hand systemic Candida mycosis in immunosuppressed patients may only lead to minor changes in antibody activities. Such situations make critical interpretation of serological findings necessary. In addition, systemic Candida-infections may not cause typical symptoms. Currently it is not possible to conclude that the results of different test systems for anti- Candida antibody detection are comparable or even exchangeable. Specificity of detected antibodies significantly depends on the test system (ELISA or HAT) or on the antigen preparation used. The detection of IgM antibodies with HAT is better than the IgG detection due to higher agglutination properties of IgM molecules. HAT mainly detects antibodies against cell wall antigens of yeast-like fungi which makes serological interpretation even more complex but gives the opportunity for differentiation. Changes in antibody concentrations may be detectable with ELISA but not with HAT, making a combination of the two techniques advantageous, e.g. in case of decreasing IgM antibody activity and simultaneously increasing IgG antibody activity. Currently no single technique in isolation allows for a definitive serological diagnosis of candidiasis. Surveillance of at risk patients and therapy control requires the use of a variety of methods including serology and antigen detection. The SERION ELISA antigen Candida test is of particular help in such situations by detecting Candida specific antigen in patient samples. Pos: 7 /Arbeitsanleitungen ELISA antigen/gültig für alle Dokumente/ELISA antigen/testprinzip/testprinzip ELISA 1 3 SERION ELISA antigen - TEST PRINCIPLE The ELISA (Enzyme Linked Immunosorbent Assay) is an immunoassay, which is particularly suited to the determination of antibodies and antigens 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 antigen microtiter plate are coated with specific antibodies directed against the pathogen of interest. If antigens in the patient s sample are present, they bind to the fixed antibody. A secondary antibody, which has been conjugated with the enzyme peroxidase, detects and binds to the immune complex. The colourless substrate hydrogen peroxide (H 2 O 2 ) and the chromogen tetramethylbenzidine (TMB) are converted into a blue coloured product. Addition of stopping solution changes the colour to yellow. The signal intensity of this reaction product is proportional to the concentration of the antigen in the sample and is measured photometrically. english 4

6 4 KIT COMPONENTS EN Test Components Pieces / Volume Break apart microtiter test strips each with eight antibody coated single wells, (altogether 96) MTP, 1 frame. 12 pieces Standard serum STD, Candida albicans antigen in human serum supplemented with fetal calf serum (FCS); negative for anti-hiv Ab, HBs-Ag (Hepatitis B-Virus surface antigen) and anti-hcv Ab; preservative: < 0.1 % sodium azide. Negative control serum NEG, Human serum supplemented with fetal calf serum (FCS); negative for anti-hiv Ab, HBs-Ag (Hepatitis B-Virus surface antigen) and anti-hcv Ab; preservative: < 0.1 % sodium azide. Anti-Candida albicans conjugate (ready-to-use) PODCR, Anti-Candida albicans antibody conjugated to horseradish-peroxidase; stabilised with protein stabilisation solution containing detergent; preservative: 0.1 % ProClin 300. Washing solution concentrate (sufficient for 1500 ml) WASH, TRIS-buffered salt solution with Tween 20; 30-fold concentrated; preservative: < 0.1 % ProClin 300. Dilution buffer SAMB, Acid solution without preservative. Stopping solution STOP, 0.5 N sulfuric acid. TMB-substrate solution (ready-to-use) TMB, TMB-hydrogen peroxide substrate solution; preservative: < 0.01 % Kathon CG. Quality control certificate with standard curve and evaluation table INFO, (quantification of antigens in U/ml). 3 x 3 ml 2 x 3 ml 13 ml 2 x 25 ml 15 ml 13 ml 13 ml 2 pieces Pos: 9 /Arbeitsanleitungen ELISA antigen/gültig für alle Dokumente/ELISA antigen/zusätzlich benötigte Materialien/Zusätzlich benötigte 1 english 5

7 5 MATERIAL REQUIRED BUT NOT SUPPLIED - common laboratory equipment - photometer for microtiter plates with filter, wavelength 450 nm, recommended reference wavelength 620 nm nm (e.g. 650 nm) - incubator 37 C - heating block 110 C - moist chamber - distilled water - Click-Clips (order no. VT120) Pos: 10 /Arbeitsanleitungen ELISA antigen/gültig für alle Dokumente/ELISA antigen/lagerung und Haltbarkeit/Lagerung und 1 english 6

8 Pos: 11 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testdurchführ ung/ü berschrift: D 1 6 STORAGE AND STABILITY EN Reagent Storage Stability Microtiter strips (coated with antibody) unopened see expiry date; Control sera / Standard sera after opening at 2 8 C in closed aluminum bag with desiccant Strips which are not used must be stored dry and air tight in the closed aluminum bag. after opening at 2 8 C Conjugate ready-to-use solution at 2 8 C Dilution buffer Avoid contamination e.g. by using sterile tips. unopened after opening at 2 8 C Discard cloudy solutions. Washing solution Concentrate after opening at 2 8 C TMB substrate working dilution at 2 8 C working dilution at room temperature Bottles used for the working dilution should be cleaned regularly. Discard cloudy solutions. ready-to-use solution at 2 8 C, stored protected from light Avoid contamination e.g. by using sterile tips. Discard if solution turns blue (extinction at 650 nm against aqua dest. > 0.2 OD). minimum shelf-life: 4 weeks shelf-life in case of proper use and storage: until expiry date see expiry date; 24 months as of date of production see expiry date; 18 months as of date of production see expiry date; 24 months as of date of production 6 months see expiry date; 24 months as of date of production 2 weeks 1 week see expiry date; 24 months as of date of production Stopping solution After opening at room temperature see expiry date english 7

9 Pos: 12 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testdurchführ ung/allgemei ne Hinweise ELISA antig 2 7 TEST PROCEDURE SERION ELISA antigen 7.1 General Notes Only use SERION ELISA antigen reagents when using SERION ELISA antigen immunoassays. The components must not be exchanged for reagents of other manufacturers. Standard and control sera as well as the conjugate of SERION ELISA antigen 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 antigen immunoassays irrespective of the lot and the test. Unopened, all components of the SERION ELISA antigen 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 aluminium bag of the microtiter plate please cut off the top of the marked side only, in order to guarantee proper resealing. Do not use the strips if the aluminium bag is damaged or if the bag with remaining strips and desiccant was not properly resealed. 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. In particular, the TMB-substrate solution is sensitive to oxidising substances and heavy metal ions. It must be stored away from light at all times and only opened in low light conditions immediately prior to use. This solution is coloured light blue-green. Skin contact with substrate and stop solution should be avoided. 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 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 antigen immunoassay is only valid if the lot-specific validation criteria on the quality control certificate are fulfilled. english 8

10 Pos: 13 /Ar bei tsanl eitungen ELISA antigen/gültig für nur ein Dokument/T estdurchführung/pr obenvor ber eitung und Lagerung/Candida antigen: Probenvorbereitung und Lager 2 Pos: 14 /Ar bei tsanl eitungen ELISA antigen/gültig für nur ein Dokument/T estdurchführung/pr obenver dünnung/candida antigen: 3 Pos: 16 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testdurchführ ung/r eagenzienvorbereitung ELISA 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! EN Take care not to damage the inscription (pathogen / AG) on the microtiter test strips during washing and aspiration to avoid confusion. 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 Sample Preparation Before running the test, specimens (patient samples, standard and negative control) (V 1 ) must be diluted in dilution buffer (V 2 ) as follows: V 1 + V 2 = 3+1 add 300 µl sample (V 1 ) each to 100 µl dilution buffer (V 2 ) After dilution and before pipetting into the microtiter plate, the samples must be mixed thoroughly to prepare a homogenous solution. Finally, the samples must be heated at 110 C (+/- 5 C) for 10 minutes. It is essential to keep both the time and temperature stated! Standard reagent vessels are not suitable for this thermolysis step and we recommend the use of safe-lock reaction vessels or similar vessels with a screw closure. Usage of a heating block thermostat requires controlling of the actual temperature by a calibrated thermometer. Consider the device-specific pre-heating period of the heating block. If necessary, the heating block must be adjusted to the required temperature. During thermolysis, a white cloudy precipitate will form and should be removed by centrifugation in a pre-cooled (4 C) table top centrifuge at 10,000 g for 10 minutes. The clear supernatant can then be used in the test. Pos: 15 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testdurchführ ung/probenl agerung ELISA antig Sample Storage The treated patient s samples should not be stored for more than 24 hours at 2 8 C. Untreated samples should not be stored at 2 8 C for more than 7 days. Extended storage is possible at -20 C. Avoid repeated freezing and thawing of samples. english 9

11 Pos: 17 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testdurchführ ung/t establauf/überschrift T 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 must be diluted with sample buffer and subsequently denatured. 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 Conjugate (ready-to-use) Avoid contamination of the ready-to-use conjugate, e.g. by using sterile tips Wash 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 ) 25 ml 750 ml 1 ml 30 ml TMB Substrate (ready-to-use) Avoid contamination of the ready-to-use substrate solution, e.g. by using sterile tips. The TMB substrate is coloured light blue-green and any solutions which exhibit a strong colour (extinction at 650 nm against distilled water of >0.2 OD) should be discarded Stopping Solution (ready-to-use) english 10

12 Pos: 18 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testdurchführ ung/t establauf/testablauf Candida Pos: 19 /Ar bei tsanl eitungen ELISA antigen/gültig für nur ein Dokument/T estdurchführung/testdurchführ ung/c andi da antigen: Manuelle Testdurchführ Overview - Test Procedure EN SERION ELISA antigen Candida quantitativ Preparation of samples, control and standard serum Sample dilution (Patient samples, control and standard serum) 300 µl Sample µl Dilution buffer INCUBATION 10 min. / 110 C CENTRIFUGATION 10 min. / 10,000 g / 4 C Retain supernatant, discard pellet Add supernatant from samples (100 µl) INCUBATION 60 min. / 37 C moist chamber WASH (5 x 300 µl DIL WASH ) Pipette conjugate solution PODCR (100 µl) INCUBATION 60 min. / 37 C moist chamber WASH (5 x 300 µl DIL WASH ) Pipette substrate solution TMB (100 µl) INCUBATION 30 min. / 37 C moist chamber Pipette stop solution STOP (100 µl) READ EXTINCTION at 450 nm (ref. 650 nm) english 11

13 Pos: 20 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testdurchführ ung/automatische T Manual Test Procedure 1. Pretreatment of samples (patient samples, standard and control serum) as described. 2. Place the required number of cavities in the frame and prepare a protocol sheet. 3. Add each 100 µl of the supernatants from the standard serum (in duplicate), negative control and patient samples into the appropriate wells of microtiter test strips. Spare one well for substrate blank, e.g.: Candida antigen quantitative cavity A1 cavity B1 cavity C1 cavity D1 Substrate blank Negative control Standard serum Standard serum cavity E1 Patient sample Sample incubation for 60 minutes (+/- 3 min.) at 37 C (+/- 1 C) in moist chamber. 5. 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 4 times (altogether 5 times!) - dry by tapping the microtiter plate on a paper towel 6. Addition of conjugate Add 100 µl of the ready-to-use conjugate into the appropriate wells (except substrate blank). 7. Conjugate incubation for 60 minutes (+/- 3 min.) at 37 C (+/- 1 C) in moist chamber. 8. After incubation wash all wells with washing solution (see above). 9. Addition of substrate Add 100 µl of ready-to-use TMB-substrate solution into each well (including well for substrate blank!). 10. Substrate incubation for 30 minutes (+/- 1 min.) at 37 C (+/- 1 C), protected from light in moist chamber. 11. Stopping of the reaction Add 100 µl stopping solution into each well. Agitate the microtiter plate gently to mix. 12. Read extinction Read optical density (OD) within 60 minutes at 450 nm against substrate blank, reference wavelength between 620 nm and 690 nm (e.g. 650 nm). english 12

14 Pos: 21 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testdurchführ ung/positi vkontrolle / Richtig 2 Pos: 22 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testauswertung/kapitelüberschrift: T ESTAU SWERTUN G Automated Test Procedure EN SERION ELISA are suited for processing on automats and evaluated for use with Immunomat TM and Gemini. 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 antigen test runs. The use of SERION ELISA controls is described in specific instruction manuals. english 13

15 Pos: 24 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testauswertung/t estg ültig keitskriteri 2 8 TEST EVALUATION SERION ELISA antigen Candida Pos: 23 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testauswertung/t estauswertung: Ein- Punkt- Quantifizier 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 antigen concentrations with the SERION ELISA antigen 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 antigen concentrations a lot specific standard curve as well as a lot specific evaluation table is included with each SERION ELISA antigen 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 antigen concentration is ensured. english 14

16 Pos: 25 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testauswertung/auswertung: Ü 2 Pos: 26 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testauswertung/nichtautomatisi erte Auswertung ELISA 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 antigen 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 antigen Candida Non-automated Evaluation For the SERION ELISA antigen test evaluation a lot-specific quality control certificate with standard curve and an evaluation table is included in the test kit. The substrate blank must be substracted from all OD values prior to evaluation. antigen/testauswertung/testauswertung 11\mod_ _18.doc Pos: 27 /Arbeitsanleitungen Methoden alle Dokumente/ELISA 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 OD, the range of the cut-off is in between OD. Method 2: Continuous Determination of Antigen 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 15

17 Pos: 28 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testauswertung/automatische 3 Pos: 29 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testauswertung/quantifizi 2 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 samples are multiplied by F. 4. Antigen 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, antigen activities are calculated online from processed and measured SERION ELISA antigen test runs by the evaluation software SERION evaluate. If the optical density of the standard is out of the valid 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 such 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 U/ml Evaluation english 16

18 Pos: 30 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa 2 Pos: 31 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testauswertung/kapitelüberschrift: Inter pretati on der 2 Pos: 33 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/testauswertung/kapitelüberschrift: R eferenzber eich gesunder Pr 2 Pos: 35 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/leistungsmer kmal e/kapitelüberschrift Leistungsmer Limits of Quantification EN The limits of quantification are specified on the quality control certificate of the SERION ELISA antigen 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 antigen activity must be multiplied by the additional dilution factor. 8.5 Borderline Range The borderline range of the SERION ELISA antigen Candida test is specified on the quality control certificate and indicates 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). 8.6 Interpretation of Results Pos: 32 /Ar bei tsanl eitungen ELISA antigen/gültig für nur ein Dokument/T estauswertung/candi da antigen/candi da antig en: Interpr etation der Ergebni Positive test results for patient samples indicate a Candida fungemia. Results can only be interpreted in combination with the clinical picture and other detection methods. A result can be considered as positive when at least 2.6 units are measured in the test. Such a result indicates that the sample contains the quantity of Mannan that is associated with 2.6 ng of Candida protein. However, it cannot distinguish between live or dead organisms, respectively. Negative test results do not exclude an acute infection, especially if high antibody titers are found. In such cases it may be that the antigen is completely masked by antibodies and even the denaturing procedure during sample preparation is not sufficient to allow detection of the antigen. In addition, the point in time during an infection when a sample is obtained, an unsuitable sample, poor sample preparation and storage can all lead to a negative result. 8.7 Reference Range of healthy Individuals Pos: 34 /Ar bei tsanl eitungen ELISA antigen/gültig für nur ein Dokument/T estauswertung/candi da antigen/candi da antig en: R efer enzbereich g esunder Testing of random blood donor sera, collected in the region of southern Germany, with the SERION ELISA antigen Candida test resulted in the following distribution. From 103 sera tested with the SERION ELISA antigen Candida test, 92 (89.3 %) were negative, six sera (5.8 %) gave a positive result and five samples (4.9 %) were determined to be borderline. english 17

19 Pos: 36 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/leistungsmer kmal e/kapitelüberschrift: Sensiti vi tät und 2 Pos: 38 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/leistungsmer kmal e/kapitelüberschrift Pr äzisi 2 9 PERFORMANCE CHARACTERISTICS 9.1 Sensitivity and Specificity Pos: 37 /Ar bei tsanl eitungen ELISA antigen/gültig für nur ein Dokument/Leistungsmer kmale/candi da antig en/candi da antig en: Sensiti vität und The SERION ELISA antigen Candida test was validated by the analysis of 148 serum samples from blood donors and 93 specimens from patients, who showed signs of candidiasis during intensive care treatment, in comparison to the results obtained with a commercially avaliable assay of a leading European manufacturer. Sera classified as borderline were not included in the calculation of sensitivity and specificity values. Performance Characteristics Sensitivity Specificity SERION ELISA antigen Candida > 99.9 % 97.8 % In another internal study, cross-reactivity with the species Candida guillermondii, Candida glabrata, Candida krusei, Candida parapsilosis and Candida tropicalis with the SERION ELISA antigen Candida was demonstrated in order to guarantee a comprehensive Candida diagnosis. Due to a similiarity of mannan with hydroxyethylstarch (HES), which is used in plasma expanders for treatment of circulatory failure (hypovolemic, hemorrhagic and septic shock), potential cross-reactivity was assessed. However, no cross-reaction with HES was measureable when using the SERION ELISA antigen Candida test. 9.2 Reproducibility Pos: 39 /Ar bei tsanl eitungen ELISA antigen/gültig für nur ein Dokument/Leistungsmer kmale/candi da antig en/candi da antig en: Pr 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 antigen Candida: Sample Mean Value Intraassay Mean Value Interassay (OD) (CV %) (OD) (CV %) negative borderline positive positive english 18

20 Pos: 40 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/allgemeine T exte ELISA 122 Pos: 41 /Ar bei tsanl eitungen ELISA antigen/gültig für alle D okumente/elisa antigen/liter atur /Kapi tel überschrift: 1 EN 10 SAFETY MEASURES 10.1 Statements of Warning The SERION ELISA antigen 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 Disposal Please observe the relevant statutory requirements! english 19

21 === Ende der Liste für T extmar ke Inhalt === 11 REFERENCES Pos: 42 /Ar bei tsanl eitungen ELISA antigen/gültig für nur ein Dokument/Liter atur/c andida antigen: [1] Fegeler, W. (1992) Möglichkeiten einer differenzierten Candida-Serologie. Pilzdialog 4, [2] Fegeler, W., Kipp, F. (2004) Candida- und Aspergillus-Antikörper-ELISA Erwartungs-werte positiver Antikörpernachweise aus Seren der klinischmykologischen Routine-diagnostik. Mycoses 47, [3] Fukazawa, Y. (1989) Antigenic structure of Candida albicans. Immunochemical basis of the serologic specificity of the mannans in yeasts. Immunol. Serol. 47, [4] Jones, J.M. (1990) Laboratory Diagnosis of Invasive Candidiasis. Clin. Microbiol. Rev. 3, [5] Matthews, R.C., Burnie, J.P., Tabaqchali, S. (1987) Isolation of Immunodominant Antigens from Sera of Patient with Systemic Candidiasis and Characterization of Serological Response to Candida albicans. J. Clin. Microbiol. 25, [6] Müllensiefen, M., Ringelmann, R. (1991) Labor-Diagnostik systemischer Candidosen. Lab. med. 15, [7] Repentigny, L. (1989) Serological Techniques for Diagnosis of Fungal Infections. Eur. J. Clin. Microbio. Infec. 8, [8] Robert Koch-Institut (2008) Epidemiologisches Bulletin 29. [9] Ruhnke, M., Rosseau, S., Graf, B. (2004) Invasive Pilzinfektionen auf der Intensivstationen. Arzneimitteltherapie 22, [10] Rüchel, R., Debusmann, F. (2008) Serologie in der Diagnostik von Mykosen. Chemother. J. 17, [11] Sendid, B., Poirot, J.L., Tabouret, M., Bonnin, A., Caillot, D., Camus, D., Poulain, D. (2002) Combined detection of mannanaemia and antimannan antibodies as a strategy for the diagnosis of systemic infection caused by pathogenic Candida species. Med. Microbiol. 51, [12] Sendid, B. et al. (2006) Candidaemia and antifungal therapy in a French University Hospital: rough trends over a decade and possible links. Infect. Dis. 6, 80. [13] Tietz, H.-J., Tausch, I. (1993) Differenzierte Candidaserologie auf dem Prüfstand. Pilzdialog 4, [14] Walsh, T.J. et al. (1991) Detection of Circulating Candida Enolase by Immunoassay in Patients with Cancer and Invasive Candidiasis. New Engl. J. Med. 324, [15] Werle, E., Kappe, R., Fiehn, W., Sonntag, H.-G. (1994) Nachweis von Anti- Candida-Antikörpern der Klassen IgM, IgG und IgA mittels Enymimmunoassays in sequentiellen Serumproben hospitalisierter Patienten. Mycoses 36, [16] Zöller, L., Krämer, I., Kappe, R., Sonntag, H.G. (1991) Enzyme Immuno Assay for Invasive Candida Infections: Reactivity of somatic Antigens of Candida albicans. J. Clin. Microbiol. 29, english 20

22 SERION ELISA antigen Candida CONTENIDO 1 USO PREVISTO 2 PERTINENCIA DIAGNÓSTICA ES 3 SERION ELISA antigen PRINCIPIO DE LA PRUEBA 4 COMPONENTES DEL KIT 5 MATERIAL NECESARIO PERO NO SUMINISTRADO 6 CONSERVACIÓN Y ESTABILIDAD 7 PROCEDIMIENTO DE LA PRUEBA SERION ELISA antigen 7.1 Notas generales 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 de pruebas 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 antigen Candida 8.4 Límites de cuantificación 8.5 Intervalo dudoso 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 1.12/09-1 Versión previa: --

23 Pos: 1 /Arbeitsanl eitung en ELISA antigen/gültig für alle Dokumente/ELISA antigen/allgemei ne Texte ELISA antigen/einl eitung " Enzymimmunoassay" C andi da Pos: 3 /Arbeitsanl eitung en ELISA antigen/gültig für alle Dokumente/ELISA antigen/allgemei ne Texte ELISA antigen/kapitelüberschrift " Anwendungsber 1 Pos: 5 /Arbeitsanl eitung en ELISA antigen/gültig für alle Dokumente/ELISA antigen/allgemei ne Texte ELISA antigen/kapitelüberschrift "Diag nostische 1 SERION ELISA antigen Candida Enzimoinmunoensayo para la determinación de antígeno de Candida Para uso diagnóstico in vitro Pos: 2 /Arbeitsanl eitung en ELISA antigen/gültig für nur ei n D okument/bestellnummer n/c andida antigen: Bestell SERION ELISA antigen Candida Nº de pedido: ESR200 1 USO PREVISTO Pos: 4 /Arbeitsanl eitung en ELISA antigen/gültig für nur ei n D okument/anwendungsbereich/c andi da antigen: SERION ELISA antigen Candida es un inmunoensayo cuantitativo y cualitativo para la detección de antígeno de Candida en el suero humano o plasma. El ensayo está recomendado para la detección de candidiasis sistémica. 2 PERTINENCIA DIAGNÓSTICA Pos: 6 /Arbeitsanl eitung en ELISA antigen/gültig für nur ei n D okument/di agnostische Bedeutung/Candida antigen: Diag nostische albicans es un hongo ubicuo que, como todas las especies de Candida, pertenece a la familia de los hongos de tipo de levadura. Dejando aparte la forma de hongo que causa principalmente infecciones superficiales, los así llamados pseudomicelios son otra manifestación morfológica de los hongos de tipo levadura. En casos de micosis sistémica aparecen principalmente tubos germinales y el desarrollo de pseudomicelios. Candida produce y excreta una serie de enzimas destructoras que capacitan al posible microorganismo patógeno para penetrar en los vasos sanguíneos y las barreras de las mucosas. Las especies Candida ssp. se transmiten principalmente mediante contaminación por suciedad de una persona a otra, siendo la principal vía de entrada la cavidad oral. Los cambios en las propiedades fungistáticas de la piel, que son una consecuencia del valor ph ligeramente ácido y de la flora bacteriana antagonista, pueden facilitar el establecimiento de candidiasis superficial en la superficie de la piel. La micosis sistémica es el resultado de la colonización de las membranas mucosas y en particular del tracto gastrointestinal. Candida ssp. puede adherirse a los epitelios de una gama de membranas mucosas mediante proteínas de adherencia y a otras estructuras de la superficie celular. El esquema a continuación muestra los pasos hipotéticos que pueden conducir a infecciones diseminadas en caso de condiciones subyacentes severas. Una descripción exacta de los diversos estadíos no es práctica (figura 1). español 2

24 Colonización del tracto nasofaríngeo Paso al lumen estomacal transferencia al lumen intestinal entrada en la circulación sanguínea transferencia al sistema linfático persopción de células de cándida ES invasión de múltiples órganos Figura 1: progresión de infecciones por cándida La candidiasis se puede clasificar por lo general en dos grupos principales cuyas características más importantes se enumeran en la tabla a continuación. superficial infecciones por Cándida micosis profunda (invasiva) localización piel, mucosas órganos principales inmunocompetencia del hospedador factores de riesgo progresión clínica + +,-/- embarazo diabetes SIDA disfunción de la piel deficiente higiene bucal inocuo en la mayoría de los casos, no es potencialmente mortal quimioterapia inmunosupresión iatrogénica catéter venoso central radioterapia quemaduras de alto grado cirugía potencialmente mortal en la mayoría de los casos, tratamiento tratamiento eficaz la eficacia del tratamiento depende del estadío de la infección Tabla 1: Características de las infecciones por Cándida español 3

25 El diagnóstico de una candidiasis basándose en métodos serológicos no es fácil en absoluto. Por un lado la colonización pasajera por el hongo puede inducir una respuesta de anticuerpos, mientras que, por otro lado, la micosis sistémica por Candida en pacientes inmunodeprimidos puede dar como resultado únicamente cambios mínimos en las actividades de los anticuerpos. Tales situaciones hacen necesaria la interpretación crítica de los resultados serológicos. Además, las infecciones sistémicas por Candida pueden no producir síntomas típicos. Actualmente no es posible concluir que los resultados de diferentes sistemas de pruebas para la detección de anticuerpos de Candida sean comparables o siquiera intercambiables. La especificidad de los anticuerpos detectados depende significativamente del sistema de pruebas (ELISA o HAT) o de la preparación de antígenos utilizados. La detección de anticuerpos IgM con HAT es mejor que la detección de IgG debido a las propiedades de aglutinación mayores de las moléculas de IgM. HAT detecta principalmente anticuerpos frente a antígenos de la pared celular de hongos de tipo levadura lo que hace la interpretación serológica aún más compleja pero proporciona la oportunidad de una diferenciación. Los cambios en las concentraciones de anticuerpos pueden ser detectables con ELISA pero no con HAT, haciendo ventajosa una combinación de ambas técnicas, p.ej. en caso de disminución de la actividad de anticuerpo IgM y simultáneamente aumento de la actividad de anticuerpo IgG. En la actualidad no existe una técnica única en aislamiento para un diagnóstico serológico definitivo de la candidiasis. La vigilancia de los pacientes en riesgo y el control de la terapia requieren el uso de diversos métodos, incluida la serología y la detección de antígenos. La prueba SERION ELISA antigen Candida resulta de particular ayuda en tales situaciones gracias a la detección de antígenos específicos de Candida en muestras de pacientes. Pos: antigen/testprinzip/testprinzip 1 ELISA ELISA für 8\mod_ _28.doc alle 3 SERION ELISA antigen PRINCIPIO DE LA PRUEBA El ELISA (Enzyme Linked Immunosorbent Assay) es un inmunoensayo, que es particularmente apropiado para la determinación de anticuerpos y antígenos en el campo de la serología de las enfermedades infecciosas. La reacción está basada en la interacción específica de los anticuerpos con su antígeno correspondiente. Las tiras de prueba de la placa de microtitulación SERION ELISA antigen están recubiertas con anticuerpos específicos dirigidos frente al patógeno de interés. Si hay presencia de antígenos en la muestra del paciente, se unen al anticuerpo fijado. Un anticuerpo secundario, que ha sido conjugado con la enzima peroxidasa, detecta el complejo inmune y se une a éste. El sustrato incoloro del peróxido de hidrógeno (H 2 O 2 ) y el cromógeno tetrametilbenzidina (TMB) se convierten en un producto de color azul. La adición de solución de parada cambia el color a amarillo. La intensidad de la señal de este producto de reacción es proporcional a la concentración del antígeno en la muestra y se mide por fotometría. Zusammensetzung/Candida 11\mod_ _28.doc Pos: 8 /Arbeitsanleitungen ELISA antigen/gültig 1 und Zusammensetzung für nur ein und español 4

26 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 anticuerpo (En total son 96) MTP, 1 bastidor. 12 partes ES Suero patrón STD, Antígeno de Candida albicans en suero humano complementado con suero fetal de ternera (FCS) 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 %. Suero control negativo NEG, Suero humano complementado con suero fetal de ternera (FCS) 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 %. Conjugado anti-candida albicans (listo para usar) PODCR, Conjugado de anticuerpo anti-candida albicans con peroxidasa de rábano picante; estabilizado con solución de estabilización de proteínas que contiene detergente; conservante: ProClin 300 0,1 %. Concentrado de dilución de lavado (suficiente para 1500 ml) WASH, Solución salina tamponada TRIS con Tween 20, concentración 30x conservante: ProClin 300 < 0,1 %. Solución amortiguadora de dilución SAMB, Solución ácida sin conservantes. Solución de parada STOP, Ácido sulfúrico 0,5 N. Solución sustrato TMB (listo para usar) TMB, Solución sustrato peróxido de hidrógeno TMB, conservante: Kathon CG < 0,01 %. Certificado de control de calidad con curva patrón y tabla de evaluación INFO, (cuantificación de antígenos en U/ml). 3 x 3 ml 2 x 3 ml 13 ml 2 x 25 ml 15 ml 13 ml 13 ml 2 partes Pos: 9 /Arbeitsanleitungen ELISA antigen/gültig für alle Dokumente/ELISA antigen/zusätzlich benötigte Materialien/Zusätzlich benötigte 1 español 5