Amersham IL-13 Human, Biotrak Easy ELISA

Transcription

1 GE Healthcare Amersham IL-13 Human, Biotrak Easy ELISA Enzyme-linked immunosorbent assay for quantitative detection of human IL-13. Product Booklet Code: RPN5972

2 Page finder 1. Legal 3 2. Handling Safety warnings and precautions Storage Expiry 4 3. Reagents provided 5 4. Intended use 6 5. Background 7 6. Principles of the test 9 7. Specimen collection Materials required but not provided Precautions for use Preparation of reagents Test protocol Calculation of results Limitations Performance characteristics Bibliography Reagent preparation summary Test protocol summary (RPN5972 IL-13) Related products 28 2

3 1. Legal GE, imagination at work and GE monogram are trademarks of General Electric Company. Amersham and Biotrak are trademarks of GE Healthcare companies. Manufactured under the proprietary technology of Bender MedSystems GmbH General Electric Company All rights reserved. Previously published 2003 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare UK Limited. Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK 3

4 2. Handling 2.1. Safety warnings and precautions Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. 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 statement(s) for specific advice Storage Store ELISA plate or whole kit at -20 C. The plate can also be removed, stored at -20 C, remaining kit reagents stored between 2 8 C Expiry 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. 4

5 3. Reagents provided 1 aluminium pouch with a Microwell Plate coated with Monoclonal Antibody (murine) to human IL-13, Biotin-Conjugate (anti-il-13 monoclonal antibody), Streptavidin-HRP and Sample Diluent, lyophilized. 2 aluminium pouches with a IL-13 Standard curve (colored). 1 bottle (25 ml) Wash Buffer Concentrate 20 (PBS with 1% Tween 20)*. 1 vial (15 ml) Substrate Solution (tetramethyl-benzidine). 1 vial (12 ml) Sample Diluent. 1 vial (15 ml) Stop Solution (1 M Phosphoric acid). 1 adhesive Plate Cover. * reagents contain preservative 5

6 4. Intended use The IL-13 Biotrak Easy ELISA is an enzyme-linked immunosorbent assay for quantitative detection of human Interleukin-13 in cell culture supernatants, human serum, plasma or other body fluids. The IL-13 Biotrak Easy ELISA is for research use only. Not for use in diagnostic or therapeutic procedures. 6

7 5. Background Interleukin-13 (IL-13) was first described as a protein product, designated P600, encoded by an RNA produced by activated mouse Th2 cells [1]. More recently, the cdna cloning of the human homologue of P600, human IL-13, has been reported [5, 6]. Human IL-13 is a nonglycosylated protein of 132 amino acids with a molecular weight of Da. The IL-13 gene is located on chromosome 5q23-31, in the same region as the genes encoding IL-3, IL-4, IL-5, and GM-CSF [9]. It is produced by activated CD4 + and CD8 + T cells, but the expression seems to be more abundant in CD4 + T cells [5]. Although it is a Th2 cytokine in the mouse, it appears to be produced by Th0, Th1, and Th2-like human T cell clones [9]. IL-13 is a pleiotropic cytokine whose spectrum of action encompasses B cells, mononuclear phagocytes, and large granular lymphocytes [5, 6, 8]. IL-13 induces CD23 expression on B cells, promotes B cell proliferation in combination with anti-ig or CD40 antibodies, and stimulates secretion of IgE and IgG4 [2, 5, 8]. IL-13 has also been shown to prolong survival of human monocytes and increases surface expression of MHC class II, CD23, and members of the integrin superfamily, like CD11b, CD11c, CD18, CD29 and CD49e [4, 5]. IL-13 inhibits the production of a series of cytokines like IL-1, IL-6, TNF-α, and IL-8 by activated human monocytes [4]. IL-13 induces IFN-γ production by NK cells [6]. The capacity of IL-13 to induce IgE synthesis indicates that IL-13 may play an important regulatory role in IgE-mediated atopic diseases [3, 8]. The measurement of IL-13 in body fluids may thus provide further information about the pathophysiology of atopic diseases. 7

8 Furthermore, IL-13 inhibits HIV-1 replication in primary culturederived macrophages and represents a candidate cytokine for the suppression of HIV infection within monocytes and macrophages in vivo [7]. 8

9 6. Principles of the test An anti-il-13 monoclonal coating antibody is adsorbed onto microwells. IL-13 present in the sample or standard binds to antibodies adsorbed to the microwells; a biotin-conjugated monoclonal anti-il-13 antibody binds to IL-13 captured by the first antibody. Streptavidin-HRP binds to the biotin conjugated anti-il-13. Following incubation unbound biotin conjugated anti IL-13 and Streptavidin-HRP is removed during a wash step, and substrate solution reactive with HRP is added to the wells. A colored product is formed in proportion to the amount of IL-13 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 seven IL-13 standard dilutions and IL-13 sample concentration determined. First Incubation - Streptavidin-Conjugate - Monoclonal Coating Antibody - Biotin -Conjugate - IL-13 Second Incubation - Substrate - Reacted Substrate 9

10 7. Specimen collection Cell culture supernatants, human serum, plasma or other biological samples will be suitable for use in the assay. Remove serum 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. Clinical samples should be kept at 2 8 C and separated rapidly before storing at -20 C to avoid loss of bioactive IL-13. If samples are to be run within 24 hours, they may be stored at 2 8 C. Avoid repeated freeze-thaw cycles. For stability and suitability of samples refer to respective chapter. 10

11 8. Materials required but not provided 5 ml and 10 ml graduated pipettes. 10 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. 11

12 9. Precautions for use 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 solutions with oxidizing agents and metal. Avoid splashing or generation of aerosols. 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 substrate reagent. Glass-distilled water or deionized water must be used for reagent preparation. Substrate solutions must be at room temperature prior to use. Decontaminate and dispose of 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. 12

13 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. 13

14 10. Preparation of reagents Wash Buffer If crystals have formed in the Wash Buffer Concentrate, warm it gently until they have completely dissolved. Pour entire contents (25 ml) of the Wash Buffer Concentrate into a clean 500 ml graduated cylinder. Bring final volume to 500 ml with glass-distilled or deionized water. Mix gently to avoid foaming. The ph of the final solution should adjust to 7.4. Transfer to a clean wash bottle and store at 2 25 C. Please note that Wash Buffer is stable for 30 days. 14

15 11. Test protocol Use plate immediately after removal from -20 C! Do not wait until pellets have completely dissolved before applying samples - the binding reaction in the standard strips starts immediately after addition of water! Do not try to dissolve pellets by pipetting up and down in the wells - some parts of the pellet could stick to the tip creating high variation of results Perform the washing step with at least 400 µl of washing buffer as stated in the manual or fill the wells completely - otherwise any pellet residues sticking to the rim of the well will not be removed and create high variation of results Remove covers of the standard strips carefully in order that all the lyophilized pellets remain in the wells a. Determine the number of Microwell strips required to test the desired number of samples plus Microwell strips for blanks and standards (colored). Each sample, standard, blank, and optional control sample should be assayed in duplicate. Remove extra Microwell Strips from holder and store in foil bag with the desiccant provided at -20 C sealed tightly. Place microwell strip containing the standard curve in position A1/A2 to H1/H2 (see Fig. 1). b. Add 150 µl of distilled water in duplicate to all standard wells (A1, A2 to G1, G2). 15

16 Figure 1. Diagram depicting an example of the arrangement of blanks, standards and samples in the microwell strips: A Standard 1 Standard 1 Sample 1 Sample 1 (100 pg/ml) (100 pg/ml) B Standard 2 (50 pg/ml) C Standard 3 (25 pg/ml) D Standard 4 (12.5 pg/ml) E Standard 5 (6.25 pg/ml) F Standard 6 (3.13 pg/ml) G Standard 7 (1.56 pg/ml) Standard 2 (50 pg/ml) Standard 3 (25 pg/ml) Standard 4 (12.5 pg/ml) Standard 5 (6.25 pg/ml) Standard 6 (3.13 pg/ml) Standard 7 (1.56 pg/ml) Sample 2 Sample 2 Sample 3 Sample 3 Sample 4 Sample 4 Sample 5 Sample 5 Sample 6 Sample 6 Sample 7 Sample 7 H Blank Blank Sample 8 Sample 8 c. Add 150 µl of distilled water, in duplicate, to the blank wells. d. Add 100 µl of distilled water to the sample wells. e. Add 50 µl of each Sample, in duplicate, to the designated wells f. Cover with a Plate Cover and incubate at room temperature (18 25 C) for 3 hours on a microplate shaker at 200 rpm. g. Remove Plate Cover and empty wells. Wash the microwell strips three times with approximately 400 µl Wash Buffer per well with thorough aspiration of microwell contents between washes. Take care not to scratch the surface of the microwells. 16

17 After the last wash, empty wells and 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. h. Pipette 100 µl of TMB Substrate Solution to all wells, including the blank wells. i. Incubate the microwell strips at room temperature (18 25 C) for 10 minutes on a microplate shaker at 200 rpm. Avoid direct exposure to intense light. The point at which the substrate reaction needs to be stopped is often determined by the ELISA reader being used. Many ELISA readers record absorbance only up to 2.0 O.D. Therefore the color development within individual microwells must be watched by the person running the assay. The substrate reaction must be stopped before positive wells are no longer properly recordable. j. 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. k. 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 IL-13 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. 17

18 12. 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 IL-13 concentration on the abscissa. Draw a best fit curve through the points of the graph. To determine the concentration of circulating IL-13 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 IL-13 concentration. *Samples have been diluted 1:2, thus the concentration read from the standard curve must be multiplied by the dilution factor (x2). *Note: There is a common dilution factor for standards and samples due to the conjugate. However only the dilution factor given in this manual has to be considered for the calculation of sample concentrations. It is suggested that each testing facility establishes a control sample of known IL-13 concentration and runs this additional control with each assay. If the values obtained are not within the expected range of this control, the assay results may be invalid. A representative standard curve is shown in Figure 2. This curve cannot be used to derive test results. Every laboratory must prepare a standard curve for each group of microwell strips assayed. 18

19 Figure 2. Representative standard curve for IL-13 ELISA. IL-13 was diluted in serial two-fold steps in Sample Diluent, symbols represent the mean of three parallel titrations. Do not use this standard curve to derive test results. A standard curve must be run for each group of microwell strips assayed. ABSORPTION 450 nm IL-13 CONCENTRATION (pg/ml) 19

20 Typical data using the IL-13 ELISA Measuring wavelength: 450 nm Reference wavelength: 620 nm Standard IL-13 Concentration (pg/ml) O.D. (450 nm) Blank O.D. Mean C.V. (%)

21 13. 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 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 detergents before use. Improper or insufficient washing 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. 21

22 14. Performance characteristics A. Sensitivity The limit of detection of IL-13 defined as the analyte concentration resulting in an absorption significantly higher than that of the dilution medium (mean plus three standard deviations) was determined to be 1.5 pg/ml (mean of 6 independent assays). B. Reproducibility a. Intra-assay Reproducibility within the assay was evaluated in independent experiments. The overall intra-assay coeffiecient of variation has been calculated to be 9.0%. b. Inter-assay Assay to assay reproducibility within one laboratory was evaluated in independent experiments by two technicians. The overall inter-assay coefficient of variation has been calculated to be 12.6%. C. Spike Recovery The spike recovery was evaluated by spiking four levels of IL-13 into pooled normal human serum. Recoveries were determined in two independent experiments with 4 replicates each. Observed values showed an overall mean recovery of 80%. D. Dilution Parallelism Human serum spiked with different levels of IL-13 was assayed at four serial threefold dilutions with 4 replicates each. Experiments showed an overall mean recovery of 92%. E. Sample Stability a. Freeze-Thaw Stability Aliquots of spiked serum were stored frozen at 20 C and thawed up to 5 times, and IL-13 levels determined. There was no significant loss of IL-13 by freezing and thawing up to 5 times. 22

23 b. Storage Stability Aliquots of spiked serum were stored at -20 C, 2-8 C, room temperature (RT) and at 37 C, and the IL-13 level determined after 24 h. There was no significant loss of IL-13 immunoreactivity during storage at -20 C, 4 C, room temperature and 37 C. F. Specificity The interference of circulating factors of the immune systems was evaluated by spiking these proteins at physiologically relevant concentrations into a IL-13 positive serum. There was no detectable cross reactivity. G. Expected Serum Values There are no detectable IL-13 levels found in serum of healthy volunteers. Elevated IL-13 levels depend on the type of immunological disorder. H. Calibration This immunoassay is calibrated with highly purified recombinant IL-13, which has been evaluated against the International Reference Standard NIBSC 94/622 and has been shown to be equivalent. NIBSC 94/622 is quantitated in International Units (IU), 1 IU corresponding to 1 ng IL

24 15. Bibliography 1) Brown, K.D., Zurawski, S.M., Mosmann, T.R. and Zurawski,. (1989) A family of small inducible proteins secreted by leukocytes are members of a new superfamily that includes leukocyte and fibroblast-derived inflammatory agents, growth factors, and indicators of various activation processes. J Immunol, 142, ) Cocks, B.G., de Waal Malefyt, R., Galizzi, J.P., de Vries, J.E. and Aversa, G. (1993) IL-13 induces proliferation and differentiation of human B cells activated by the CD40 ligand. Int. Immunol, 5, ) De Vries, J.E. and Zurawski, G. (1995) Immunoregulatory properties of IL-13: Its potential role in atopic disease. Int. Arch. Allergy Immunol, 106, ) De Waal Malefyt, R., Figdor, C.G., Huijbens, R., Mohan Peterson, S., Bennett, B., Culpepper, J., Dang, W., Zurawski, G. and de Vries, J.E. (1993) Effects of IL-13 on phenotype, cytokine production, and cytotoxic function of human monocytes. Comparison with IL-4 and modulation by IFN-gamma or IL-10. J Immunol, 151, ) McKenzie, A.N., Culpepper, J.A., de Waal Malefyt, R., Briere, F., Punnonen, J., Aversa, G., Sato, A., Dang, W., Cocks, B.G., Menon, S. and et al (1993) Interleukin 13, a T-cell-derived cytokine that regulates human monocyte and B-cell function. Proc. Natl. Acad. Sci. U. S. A., 90, ) Minty, A., Chalon, P., Derocq, J.M., Dumont, X., Guillemot, J.C., Kaghad, M., Labit, C., Leplatois, P., Liauzun, P., Miloux, B. and et al (1993) Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses. Nature, 362,

25 7) Montaner, L.J., Doyle, A.G., Collin, M., Herbein, G., Illei, P., James, W., Minty, A., Caput, D., Ferrara, P. and Gordon, S. (1993) Interleukin 13 inhibits human immunodeficiency virus type 1 production in primary blood-derived human macrophages in vitro. J Exp. Med., 178, ) Punnonen, J., Aversa, G., Cocks, B.G., McKenzie, A.N., Menon, S., Zurawski, G., de Waal Malefyt, R. and de Vries, J.E. (1993) Interleukin 13 induces interleukin 4-independent IgG4 and IgE synthesis and CD23 expression by human B cells. Proc. Natl. Acad. Sci. U. S. A., 90, ) Zurawski, G. and de Vries, J.E. (1994) Interleukin 13, an interleukin 4-like cytokine that acts on monocytes and B cells, but not on T cells. Immunol Today, 15,

26 16. Reagent preparation summary Wash Buffer Add Wash Buffer Concentrate 20 (25 ml) to 475 ml distilled water. 26

27 17. Test protocol summary (RPN5972 IL-13) Place standard strip in position A1/A2 to H1/H2. Add 150 µl distilled water, in duplicate, to standard wells Add 150 µl distilled water, in duplicate, to the blank wells. Add 100 µl distilled water, in duplicate, to the sample wells. Add 50 µl sample, in duplicate, to designated wells. Cover microwell strips and incubate 3 hours at room temperature (18 25 C) on microplate shaker. 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 10 minutes at room temperature (18 25 C) on microplate shaker. Add 100 µl Stop Solution to all wells including blank wells. Blank microwell reader and measure color intensity at 450 nm. Note: *Samples have been diluted 1:2, thus the concentration read from the standard curve must be multiplied by the dilution factor ( 2). *Note: There is a common dilution factor for standards and samples due to the conjugate. However only the dilution factor given in this manual has to be considered for the calculation of sample concentrations. 27

28 17. Related products IFN-α Human Biotrak Easy ELISA IFN-γ Human Biotrak Easy ELISA IL-10 Human Biotrak Easy ELISA IL-10 Mouse Biotrak Easy ELISA MCP-1 Human Biotrak Easy ELISA IL-2 Human Biotrak Easy ELISA IL-2 Mouse Biotrak Easy ELISA TFN-α Human Biotrak Easy ELISA IL-6 Human Biotrak Easy ELISA IL-8/NAP-1 Human Biotrak Easy ELISA TGF-β1 Human Biotrak Easy ELISA IL-1β Human Biotrak Easy ELISA TFN-β Human Biotrak Easy ELISA RPN5960 RPN5961 RPN5962 RPN5963 RPN5964 RPN5965 RPN5966 RPN5967 RPN5968 RPN5969 RPN5970 RPN5971 RPN

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32 GE Healthcare offices: GE Healthcare Bio-Sciences AB Björkgatan 30, Uppsala, Sweden GE Healthcare Europe GmbH Munzinger Strasse 5, D Freiburg, Germany GE Healthcare Bio-Sciences Corp. 800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ , USA GE Healthcare Bio-Sciences KK Sanken Bldg , Hyakunincho, Shinjuku-ku, Tokyo , Japan For contact information for your local office, please visit: GE Healthcare UK Limited Amersham Place Little Chalfont, Buckinghamshire, HP7 9NA, UK imagination at work RPN5972PL Rev B 2/2008