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1 Instructions for Use sicam-2 ELISA Enzyme immunoassay (microtiter strips) for the quantitative determination of soluble Intercellular Adhesion Molecule-2 levels in cell culture supernatants, human serum, plasma, or other body fluids BE C I B L I N T E R N A T I O N A L G M B H Flughafenstrasse 52a Phone: +49 (0) IBL@IBL-International.com D Hamburg, Germany Fax: +49 (0)

2 TABLE OF CONTENTS 1. INTENDED USE 2 2. SUMMARY 2 3. PRINCIPLES OF THE TEST 4 4. REAGENTS PROVIDED 5 5. STORAGE INSTRUCTIONS 6 6. SPECIMEN COLLECTION 6 7. MATERIALS REQUIRED BUT NOT PROVIDED 7 8. PRECAUTIONS FOR USE 8 9. PREPARATION OF REAGENTS TEST PROTOCOL CALCULATION OF RESULTS LIMITATIONS PERFORMANCE CHARACTERISTICS BIBLIOGRAPHY ORDERING INFORMATION REAGENT PREPARATION SUMMARY TEST PROTOCOL SUMMARY (07)

3 2 1. INTENDED USE The sicam-2 ELISA is an enzyme-linked immunosorbent assay for the quantitative detection of soluble Intercellular Adhesion Molecule-2 levels in cell culture supernatants, human serum, plasma, or other body fluids. The ICAM-2 ELISA is for research use only. Not for use in diagnostic or therapeutic procedures. 2. SUMMARY Intercellular Adhesion Molecule-2 (ICAM-2) is a 60 kda surface glycoprotein that promotes adhesion between immune cells and the vascular endothelium. ICAM-2 is a member of the Ig-superfamily with two immunoglobulin-like extracellular domains (3). ICAM-2 is much more closely related to the two N-terminal domains of ICAM-1 (34% identity) than either ICAM-1 or ICAM-2 is to other members of the Igsuperfamily, demonstrating the existence of a subfamily of Ig-like ligands that bind the same integrin receptor (3). ICAM-2 is broadly distributed on hematopoietic cells. On resting lymphocytes, the ICAM-2 expression is several-fold higher than that seen with ICAM-1, while monocytes express equivalent levels of ICAM-1 and ICAM-2. In contrast to ICAM-1, neutrophils stain negative for ICAM-2. Just little or no ICAM- 2 staining can be demonstrated on other cell lines, the sole exception being (i) resting vascular endothelial cells, which possess high levels of ICAM-2, and (ii) small clusters of cells in lymphoid tissue germinal centres (2). ICAM-2 expression is unaffected by a variety of inflammatory cytokines, while ICAM-1 is upregulated upon stimulation. This points toward the importance of ICAM-2 in the unstimulated resting state, before ICAM-1 expression is increased (2). The integrin LFA-1 is the receptor for ICAM-2 and ICAM-1 as well (4). The functional characteristics of ICAM-2 as LFA-1 ligand can be summarized as follows:

4 3 ICAM-2 dominates over ICAM-1 on resting endothelial cells and seems to be involved in the recirculation of LFA-1 positive lymphocytes, e.g. facilitating T-memory cell recirculation. As resting T-cells express little or no ICAM-1, ICAM-2 may also be important in initial T-cell adhesion with antigen-presenting cells that bear LFA-1. Also the lysis of certain target cells appears to occur in an ICAM-1 independent manner, possibly regulated by ICAM-2 (1, 2).

5 4 3. PRINCIPLES OF THE TEST An anti-sicam-2 monoclonal coating antibody is adsorbed onto microwells. Coated Microwell sicam-2 present in the sample or standard binds to antibodies adsorbed to the microwells; a biotin-conjugated monoclonal anti-sicam-2 antibody is added and binds to sicam-2 captured by the first antibody. - Monoclonal Coating Antibody First Incubation - sicam-2 - Biotin-Conjugate Following incubation unbound biotin conjugated anti-sicam-2 is removed during a wash step. Streptavidin-HRP is added and binds to the biotin conjugated anti-sicam-2. Following incu-bation unbound Streptavidin-HRP is removed during a wash step, and substrate solution reactive with HRP is added to the wells. Second Incubation - Streptavidin-Conjugate Third Incubation A coloured product is formed in proportion to the amount of sicam-2 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450 nm. A standard curve is prepared from six sicam-2 standard dilutions and sicam- 2 sample concentration determined. - Substrate - Reacted Substrate

6 5 4. REAGENTS PROVIDED 1 aluminium pouch with a microwell plate coated with Monoclonal Antibody (murine) to human sicam-2 1 vial (0.3 ml) Biotin-Conjugate anti-sicam-2 monoclonal (murine) antibody 2 vials (5 µl) Streptavidin HRP-Concentrate 2 vials 24 U/ml sicam-2 Standard, lyophilized; 1 bottle (10 ml) Wash Buffer Concentrate 200x 1 bottle (23 ml) Streptavidin-HRP diluent 1 bottle (25 ml) Standard buffer diluent (10x) 1 bottle (7.5 ml) Biotin-Conjugate diluent 1 bottle (11 ml) Substrate Solution (tetramethyl-benzidine and buffered hydrogen peroxide) 1 vial (11 ml) Stop Solution (1.8N H 2 SO 4 ) 1 vial (0.4 ml each) Blue-Dye, Red-Dye, Green-Dye 2 adhesive Plate Covers Reagent Labels

7 6 5. STORAGE INSTRUCTIONS Store kit reagents between 2 and 8 C. Immediately after use reagents should be returned to cold storage (2 to 8 C). Expiry of the kit and reagents is stated on labels. The expiry of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, the reagent is not contaminated by the first handling. 6. SPECIMEN COLLECTION Cell culture supernatants, human serum, or other body fluids are suitable for use in the assay. Remove the serum or plasma from the clot or red cells, respectively, as soon as possible after clotting and separation. Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens. Samples must be stored frozen at -20 C to avoid loss of bioactive sicam-2. If samples are to be run within 24 hours, they may be stored at 2 to 8 C. Avoid repeated freeze-thaw cycles. Prior to assay, frozen sera or plasma should be brought to room temperature slowly and mixed gently. For sample stability refer to 13.

8 7. MATERIALS REQUIRED BUT NOT PROVIDED 7-5 ml and 10 ml graduated pipettes - 5 µl to 1000 µl adjustable single channel micropipettes with disposable tips - 50 µl to 300 µl adjustable multichannel micropipette with disposable tips - Multichannel micropipette reservoir - Beakers, flasks, cylinders necessary for preparation of reagents - Device for delivery of wash solution (multichannel wash bottle or automatic wash system) - Microwell strip reader capable of reading at 450 nm (620 nm as optional reference wave length) - Glass-distilled or deionized water - Statistical calculator with program to perform linear regression analysis

9 8 8. PRECAUTIONS FOR USE - All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety statements(s) for specific advice. - Reagents are intended for research use only and are not for use in diagnostic or therapeutic procedures. - Do not mix or substitute reagents with those from other lots or other sources. - Do not use kit reagents beyond expiration date on label. - Do not expose kit reagents to strong light during storage or incubation. - Do not pipette by mouth. - Do not eat or smoke in areas where kit reagents or samples are handled. - Avoid contact of skin or mucous membranes with kit reagents or specimens. - Rubber or disposable latex gloves should be worn while handling kit reagents or specimens. - Avoid contact of substrate solution with oxidizing agents and metal. - Avoid splashing or generation of aerosols.

10 9 - In order to avoid microbial contamination or cross-contamination of reagents or specimens which may invalidate the test use disposable pipette tips and/or pipettes. - Use clean, dedicated reagent trays for dispensing the conjugate and substrate reagents. - Exposure to acids will inactivate the conjugate. - Glass-distilled water or deionized water must be used for reagent preparation. - Substrate solution must be at room temperature prior to use. - Decontaminate and dispose specimens and all potentially contaminated materials as if they could contain infectious agents. The preferred method of decontamination is autoclaving for a minimum of 1 hour at C. - Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1.0% sodium hypochlorite. Allow 30 minutes for effective decontamination. Liquid waste containing acid must be neutralized prior to the addition of sodium hypochlorite.

11 9. PREPARATION OF REAGENTS A. Wash Buffer 10 If crystals have formed in the Wash Buffer Concentrate, warm it gently until they have completely dissolved. Pour entire contents (10 ml) of the vial of the Wash Buffer Concentrate into a clean 2,000 ml graduated cylinder. Bring final volume to 2,000 ml with glass-distilled or deionized water. Mix gently to avoid foaming. Transfer to a clean wash bottle and store at 2 to 25 C. Please note that Wash Buffer is stable for 30 days. B. Standard Buffer Diluent Before use, dilute 5 ml standard buffer diluent in 50 ml distilled water or all the content of the vial (25 ml) in 250 ml distilled water. C. Preparation of Standards Standards have to be reconstituted with standard buffer diluent. Reconstitution volume is stated on the label of the standard vial. Shake gently to ensure complete reconstitution. D. Preparation of Biotin-Conjugate Dilute the Biotin-Conjugate with the biotin-conjugate diluent in a clean glass vial according to the number of strips to be used. Number Biotin-Conjugate Biotin-conjugate of Strips (ml) Diluent (ml)

12 11 E. Preparation of Streptavidin-HRP Add 0.5 ml of Streptavidin-HRP diluent to one vial of Streptavidin- HRP-concentrate (5 µl). Do not keep this dilution for further experiments. According to the number of strips used prepare dilutions of Streptavidin- HRP with Streptavidin-HRP diluent in a clean test tube. Streptavidin- HRP may be prepared as needed according to the following table: Number Streptavidin-HRP Streptavidin-HRP of Strips (µl) Diluent (ml) F. Addition of colour-giving reagents: Blue-Dye, Green-Dye, Red-Dye In order to help our customers to avoid any mistakes in pipetting the IBL-International ELISAs, IBL-International now offers a new tool that helps to monitor the addition of even very small volumes of a solution to the reaction well by giving distinctive colours to each step of the ELISA procedure. This procedure is optional, does not in any way interfere with the test results, and is designed to help the customer with the performance of the test, but can also be omitted, just following the instruction booklet.

13 12 Alternatively, the dye solutions from the stocks provided (Blue-Dye, Green-Dye, Red-Dye) can be added to the reagents according to the following guidelines: 1. Diluent: Before sample dilution add the Blue-Dye at a dilution of 1:250 (see table below) to the appropriate diluent (1x) according to the test protocol. After addition of Blue-Dye, proceed according to the instruction booklet. 5 ml Diluent 20 µl Blue-Dye 12 ml Diluent 48 µl Blue-Dye 50 ml Diluent 200 µl Blue-Dye 60 ml Diluent 240 µl Blue-Dye 2. Biotin-Conjugate: Before dilution of the concentrated conjugate, add the Green-Dye at a dilution of 1:100 (see table below) to the Diluent used for the final conjugate dilution. Proceed after addition of Green-Dye according to the instruction booklet, preparation of Biotin-conjugate. 3 ml Diluent 30 µl Green-Dye 6 ml Diluent 60 µl Green-Dye 12 ml Diluent 120 µl Green-Dye 3. Streptavidin-HRP: Before dilution of the concentrated Streptavidin- HRP; add the Red-Dye at a dilution of 1:250 (see table below) to the Diluent used for the final Streptavidin-HRP dilution. Proceed after addition of Red-Dye according to the instruction booklet, preparation of Streptavidin-HRP. 6 ml Diluent 24 µl Red-Dye 12 ml Diluent 48 µl Red-Dye

14 TEST PROTOCOL a. Before being tested, human serum samples have to be diluted 20 times in standard buffer diluent according to the following scheme: Add 15 µl of serum sample to 285 µl standard buffer diluent b. Mix all reagents thoroughly without foaming before use. c. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Each sample, standard, blank and optional control sample should be assayed in duplicate. Remove extra Microwell Strips coated with Monoclonal Antibody (murine) to human sicam-2 from holder and store in foil bag with the desiccant provided at 2-8 C sealed tightly. d. Add 100 µl of Standard Buffer, in duplicate, to the standard wells, leaving the first wells (24 U/ml) empty. Prepare standard dilutions by pipetting 200 µl of sicam-2 Standard, in duplicate, into well A1 and A2 (see Figure 1 and 2). Transfer 100 µl to well B1 and B2 respectively. Take care not to scratch the inner surface of the microwells. Mix the contents of well B1 and B2 and transfer 100 µl to well C1 and C2 respectively. Continue this procedure three times, creating two rows of sicam-2 standard dilutions ranging from 24 to 0.75 U/ml. Discard 100 µl of the contents from the last microwells (F1, F2) used.

15 14 Figure 1. Preparation of sicam-2 standard dilutions: transfer100 µl sicam-2 Standard 200 µl A1 B1 C1 D1 - F1 100 µl Standard Buffer Diluent discard 100 µl Figure 2. Diagram depicting an example of the arrangement of blanks, standards and samples in the microwell strips: A Standard 1 Standard 1 Sample 2 Sample 2 (24 U/ml) (24 U/ml) B Standard 2 Standard 2 Sample 3 Sample 3 (12 U/ml) (12 U/ml) C Standard 3 Standard 3 Sample 4 Sample 4 (6 U/ml) (6 U/ml) D Standard 4 Standard 4 Sample 5 Sample 5 (3 U/ml) (3 U/ml) E Standard 5 Standard 5 Sample 6 Sample 6 (1.5 U/ml) (1.5 U/ml) F Standard 6 Standard 6 Sample 7 Sample 7 (0.75 U/ml) (0.75 U/ml) G Blank Blank Sample 8 Sample 8 H Sample 1 Sample 1 Sample 9 Sample 9

16 e. Add 100 µl of Standard Buffer, in duplicate, to the blank wells. 15 f. Add 100 µl of diluted samples to all wells designated for samples. g. Add 50 µl of diluted Biotin-Conjugate to all wells. (Refer to preparation of reagents) h. Cover with a Plate Cover and incubate at room temperature (18 to 25 C) for 1 hour, if available on a rotator set at 100 rpm. i. Remove Plate Cover and empty wells. Wash the microwell strips three times with approximately 300 µl Wash Buffer per well with thorough aspiration of microwell contents between washes. Take care not to scratch the surface of the microwells. After the last wash, tap microwell strips on absorbent pad or paper towel to remove excess Wash Buffer. Use the microwell strips immediately after washing or place upside down on a wet absorbent paper for not longer than 15 minutes. Do not allow wells to dry. j. Prepare Streptavidin-HRP Solution. (Refer to preparation of reagents). k. Add 100 µl of Streptavidin- HRP Solution to all wells, including the blank wells. l. Cover with a Plate Cover and incubate at room temperature (18 to 25 C) for 30 minutes, if available on a rotator set at 100 rpm. m. Remove Plate Cover and empty wells. Wash microwell strips 3 times according to point i. of the test protocol. Proceed immediately to the next step. n. Pipette 100 µl of TMB Substrate Solution to all wells, including the blank wells.

17 16 o. Incubate the microwell strips at room temperature (18 to 25 C) for about 10 minutes. Avoid direct exposure to intense light. The colour development on the plate should be monitored and the substrate reaction stopped (see point p. of this protocol) before positive wells are no longer properly recordable. It is recommended to add the stop solution when the highest standard has developed a dark blue colour. Alternatively the colour development can be monitored by the ELISA reader at 620nm. The substrate reaction should be stopped as soon as an OD of is reached. p. Stop the enzyme reaction by quickly pipetting 100 µl of Stop Solution into each well, including the blank wells. It is important that the Stop Solution is spread quickly and uniformly throughout the microwells to completely inactivate the enzyme. Results must be read immediately after the Stop Solution is added or within one hour if the microwell strips are stored at 2-8 C in the dark. q. Read absorbance of each microwell on a spectro-photometer using 450 nm as the primary wave length (optionally 620 nm as the reference wave length; 610 nm to 650 nm is acceptable). Blank the plate reader according to the manufacturer's instructions by using the blank wells. Determine the absorbance of both, the samples and the sicam-2 standards. Note: In case of incubation without shaking the obtained O.D. values may be lower than indicated below. Nevertheless the results are still valid.

18 CALCULATION OF RESULTS - Calculate the average absorbance values for each set of duplicate standards and samples. Duplicates should be within 20 per cent of the mean. - Create a standard curve by plotting the mean absorbance for each standard concentration on the ordinate against the sicam-2 concentration on the abscissa. Draw a best fit curve through the points of the graph. - To determine the concentration of circulating sicam-2 for each sample, first find the mean absorbance value on the ordinate and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding sicam-2 concentration. - For samples which have been diluted according to the instructions given in this manual 1 : 20 the concentration read from the standard curve must be multiplied by the dilution factor (x 20). Note: Calculation of samples with an O.D. exceeding 2.0 may result in incorrect low sicam-2 levels. Such samples require further dilution of 1:40-1:80 with Standard buffer Diluent in order to precisely quantitate the actual sicam- 2 level. - It is suggested that each testing facility establishes a control sample of known sicam-2 concentration and runs this additional control with each assay. If the values obtained are not within the expected range of the control, the assay results may be invalid.

19 LIMITATIONS - Since exact conditions may vary from assay to assay, a standard curve must be established for every run. - Bacterial or fungal contamination of either screen samples or reagents or cross-contamination between reagents may cause erroneous results. - Disposable pipette tips, flasks or glassware are preferred, reusable glassware must be washed and thoroughly rinsed of all detergent before use. - Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty wells before dispensing fresh Wash Buffer, fill with Wash Buffer as indicated for each wash cycle and do not allow wells to sit uncovered or dry for extended periods. - The use of radioimmunotherapy has significally increased the number of patients with human anti-mouse IgG antibody (HAMA). HAMA may interfere with assays utilizing murine monoclonal antibodies leading to both false positive and false negative results. Serum samples containing antibodies to murine immunoglobulins can still be analyzed in such assays when murine immunoglobulins (serum, ascitic fluid, or monoclonal antibodies of irrelevant specificity) are added to the Standard buffer Diluent.

20 PERFORMANCE CHARACTERISTICS A. Sensitivity The limit of detection for sicam-2, defined as the analyte concentration resulting in an absorption significantly higher than the absorption of the dilution medium (mean plus two standard deviations) was determined to be 0.30 U/ml (mean of 20 independent assays). B. Reproducibility a. Intra-assay Reproducibility within the assay was evaluated in ten independent experiments of 2 serum samples containing different concentrations of sicam-2. Two standard curves were run on each plate. The overall intra-assay coefficient of variation has been calculated to be 6.7%. b. Inter-assay Assay to assay reproducibility within one laboratory was evaluated in eighteen independent experiments by three technicians of 2 serum samples containing different concentrations of sicam-2. Two standard curves were run on each plate. The overall inter-assay coefficient of variation has been calculated to be 7.0%. C. Expected Values A panel of 80 sera from healthy blood donors (male and female) was tested for sicam-2. The detected sicam-2 levels ranged between 44 and 405 U/ml with a mean level of 220 U/ml and a standard deviation of 74 U/ml.

21 D. Sample Freeze-Thaw Stability 20 Aliquots of serum samples (unspiked or spiked with sicam-2) were stored at -20 C and thawed several times, and the sicam-2 level determined. There was no significant loss of sicam-2 concentrations between 0 and 5 freeze-thaw cycles. E. Sample Storage Stability Aliquots of a serum sample (unspiked or spiked with sicam-2) were stored at -20 C, 2-8 C, room temperature and at 37 C and the sicam-2 level determined after 24 hours. There was no significant loss of sicam- 2 immunoreactivity during storage under above conditions. F. Comparison of Serum and Plasma From three individuals, serum as well as EDTA, citrate and heparin plasma obtained at the same time point were evaluated. sicam-2 levels were not significantly different and therefore all these blood preparations are suitable for sicam-2 determinations. Nevertheless, the uniformity of samples compared is recommended. G. Specificity The interference of circulating factors of the immune system was evaluated by spiking these proteins at physiologically relevant concentrations into a sicam-2 positive serum. There was no detectable cross reactivity with any of the tested proteins, notably there was no cross reactivity with sicam-1 and sicam-3.

22 BIBLIOGRAPHY 1) De Fougerolles A. R., S. A. Stacker, R. Schwarting, and T. A. Springer. (1991). Characterization of ICAM-2 and evidence for a third counter-receptor for LFA-1. J. Exp. Med. 174, ) Gahmberg C. G., P. Nortamo, D. Zimmermann, and E. Ruoslahti. (1991). The human leukocyte-adhesion ligand, intercellularadhesion molecule 2. Expression and characterization of the protein. Eur. J. Biochem. 177, ) Seth R., R. Salcedo, M. Patarroyo, and M. W. Makgobe. (1991). ICAM-2 peptides mediate lymphocyte adhesion by binding to CD11a/CD18 and CD49d/CD29 integrins. FEBS 282, ) Staunton D. E., M. L. Dustin, and T. A. Springer. (1989). Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1. Nature 339,

23 ORDERING INFORMATION For orders please contact: See last page For technical information please contact:

24 REAGENT PREPARATION SUMMARY A. Wash Buffer Add Wash Buffer Concentrate 200 x (10 ml) in 2000 ml distilled water. B. Standard Buffer Diluent Dilute 25 ml of Standard Buffer Diluent in 250 ml of distilled water. C. Standards Standards have to be reconstituted with standard buffer diluent. Reconstitution volume is stated on the label of the standard vial. D. Biotin-Conjugate Number Biotin- Biotin- Conjugate of Strips Conjugate (ml) Diluent (ml) E. Streptavidin-HRP Add 500 µl Streptavidin-HRP diluent to vial of Streptavidin-HRP Concentrate Make further dilutions according to table: Number Streptavidin- HRP -Diluent of Strips HRP (µl) (ml)

25 17. TEST PROTOCOL SUMMARY 24 - Dilute serum samples with Standard buffer Diluent - Add 100 µl Standard buffer Diluent, in duplicate, to standard wells except the first wells (24 U/ml) - Pipette 200 µl sicam-2 Standard into the first standard wells and create standard dilutions ranging from 24 to 0.75 U/ml by transferring 100 µl from well to well; Discard 100µl from the last wells. - Add 100 µl Standard Diluent, in duplicate, to the blank wells - Add 100 µl of diluted Sample to designated wells - Prepare Biotin-Conjugate - Add 50 µl of diluted Biotin-Conjugate to all wells - Cover microwell strips and incubate 1 hour at room temperature (18 to 25 C) - Empty and wash microwell strips 3 times with Wash Buffer - Prepare Streptavidin-HRP - Add 100 µl diluted Streptavidin-HRP to all wells - Cover microwell strips and incubate 30 minutes at room temperature (18 to 25 C) - Empty and wash microwell strips 3 times with Wash Buffer - Add 100 µl of TMB Substrate Solution to all wells including blank wells - Incubate the microwell strips for about 20 minutes at room temperature (18 to 25 C) - Add 100 µl Stop Solution to all wells including blank wells - Blank microwell reader and measure colour intensity at 450 nm Note: For samples which have been diluted according to the instructions given in this manual 1:20 the concentration read from the standard curve must be multiplied by the dilution factor (x20). Calculation of samples with an O.D. exceeding 2.0 may result in incorrect low sicam-2 levels. Such samples require further dilution of 1:40-1:80 with Standard Diluent in order to precisely quantitate the actual sicam-2 level.

26 Symbols / Symbole / Symbôles / Símbolos / Símbolos / Σύµβολα REF LOT Cat.-No.: / Kat.-Nr.: / No.- Cat.: / Cat.-No.: / N.º Cat.: / N. Cat.: / Αριθµός-Κατ.: Lot-No.: / Chargen-Bez.: / No. Lot: / Lot-No.: / Lote N.º: / Lotto n.: / Αριθµός -Παραγωγή: Use by: / Verwendbar bis: / Utiliser à: / Usado por: / Usar até: / Da utilizzare entro: / Χρησιµοποιείται από: No. of Tests: / Kitgröße: / Nb. de Tests: / No. de Determ.: / N.º de Testes: / Quantità dei tests: / Αριθµός εξετάσεων: CONC Concentrate / Konzentrat / Concentré / Concentrar / Concentrado / Concentrato / Συµπύκνωµα LYO IVD Lyophilized / Lyophilisat / Lyophilisé / Liofilizado / Liofilizado / Liofilizzato / Λυοφιλιασµένο In Vitro Diagnostic Medical Device. / In-vitro-Diagnostikum. / Appareil Médical pour Diagnostics In Vitro. / Dispositivo Médico para Diagnóstico In Vitro. / Equipamento Médico de Diagnóstico In Vitro. / Dispositivo Medico Diagnostico In vitro. / Ιατρική συσκευή για In-Vitro ιάγνωση. Evaluation kit. / Nur für Leistungsbewertungszwecke. / Kit pour évaluation. / Juego de Reactivos para Evaluació. / Kit de avaliação. / Kit di evaluazione. / Κιτ Αξιολόγησης. Read instructions before use. / Arbeitsanleitung lesen. / Lire la fiche technique avant emploi. / Lea las instrucciones antes de usar. / Ler as instruções antes de usar. / Leggere le istruzioni prima dell uso. / ιαβάστε τις οδηγίες πριν την χρήση. Keep away from heat or direct sun light. / Vor Hitze und direkter Sonneneinstrahlung schützen. / Garder à l abri de la chaleur et de toute exposition lumineuse. / Manténgase alejado del calor o la luz solar directa. / Manter longe do calor ou luz solar directa. / Non esporre ai raggi solari. / Να φυλάσσεται µακριά από θερµότητα και άµεση επαφή µε το φως του ηλίου. Store at: / Lagern bei: / Stocker à: / Almacene a: / Armazenar a: / Conservare a: / Αποθήκευση στους: Manufacturer: / Hersteller: / Fabricant: / Productor: / Fabricante: / Fabbricante: / Παραγωγός: Caution! / Vorsicht! / Attention! / Precaución! / Cuidado! / Attenzione! / Προσοχή! Symbols of the kit components see MATERIALS SUPPLIED. Die Symbole der Komponenten sind im Kapitel KOMPONENTEN DES KITS beschrieben. Voir MATERIEL FOURNI pour les symbôles des composants du kit. Símbolos de los componentes del juego de reactivos, vea MATERIALES SUMINISTRADOS. Para símbolos dos componentes do kit ver MATERIAIS FORNECIDOS. Per i simboli dei componenti del kit si veda COMPONENTI DEL KIT. Για τα σύµβολα των συστατικών του κιτ συµβουλευτείτε το ΠΑΡΕΧΟΜΕΝΑ ΥΛΙΚΑ. IBL AFFILIATES WORLDWIDE IBL International GmbH Flughafenstr. 52A, Hamburg, Germany IBL International B.V. Zutphenseweg 55, 7418 AH Deventer, The Netherlands IBL International Corp. 194 Wildcat Road, Toronto, Ontario M3J 2N5, Canada Tel.: + 49 (0) Fax: IBL@IBL-International.com WEB: Tel.: + 49 (0) Fax: IBL@IBL-International.com WEB: Tel.: +1 (416) Fax: Sales@IBL-International.com WEB: LIABILITY: Complaints will be accepted in each mode written or vocal. Preferred is that the complaint is accompanied with the test performance and results. Any modification of the test procedure or exchange or mixing of components of different lots could negatively affect the results. These cases invalidate any claim for replacement. Regardless, in the event of any claim, the manufacturer s liability is not to exceed the value of the test kit. Any damage caused to the kit during transportation is not subject to the liability of the manufacturer Symbols Version 3.5 /