ab IL-2 (Interleukin 2) + IL-4 (Interleukin 4) Human SimpleStep ELISA Kit

Transcription

1 ab IL-2 (Interleukin 2) + IL-4 (Interleukin 4) Human SimpleStep ELISA Kit Instructions for Use For the simultaneous quantitative measurement of IL-2 in parallel with IL-4 in Human cell culture supernatant, plasma and serum samples. This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 29/08/2013

2 Table of Contents INTRODUCTION BACKGROUND ASSAY SUMMARY 2 4 GENERAL INFORMATION PRECAUTIONS STORAGE AND STABILITY MATERIALS SUPPLIED MATERIALS REQUIRED, NOT SUPPLIED LIMITATIONS TECHNICAL HINTS ASSAY PREPARATION REAGENT PREPARATION STANDARD PREPARATION - IL-2 STANDARD PREPARATION IL-4 SAMPLE PREPARATION PLATE PREPARATION ASSAY PROCEDURE 14. ASSAY PROCEDURE 18 DATA ANALYSIS CALCULATIONS TYPICAL DATA IL-2 TYPICAL DATA IL-4 SPECIES REACTIVITY RESOURCES TROUBLESHOOTING NOTES

3 INTRODUCTION 1. BACKGROUND Abcam s IL-2 (Interleukin-2) and IL-4 (Interleukin-4) in vitro multitarget SimpleStep ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the simultaneous, accurate and quantitative measurement of IL-2 in parallel with IL-4 in Human cell culture supernatant, plasma and serum samples. The multi-target SimpleStep ELISA measures a single target per well and employs a labeled target specific capture and target specific detector antibody which immunocaptures the sample analyte in solution. This entire complex (capture antibody/protein/detector antibody) is in turn immobilized in the well by immunoaffinity via the anti-tag antibody. Samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material then the TMB substrate is added. The addition of Stop Solution extinguishes the color development and changes the final color from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm. IL-2, also known as T cell growth factor (TCGF), is a glycosylated alpha-helical polypeptide, synthesized as a 153 amino acid (aa) precursor with a 20 aa signal peptide and a 133 aa mature chain. It is secreted by activated CD4+ and CD8+ T cells, neurons, microglia and hematopoietic stem cells in response to antigenic or mitogenic stimulation. IL-2 is required for T-cell proliferation, Natural Killer cells (NK) cytolytic activity, differentiation of regulatory T cells, modulation of T helper (Th) cell differentiation and activation-induced cell death. In particular, IL-2 modulates the expression of receptors for other cytokines and transcription factors, therefore regulating cytokine cascades that correlate with each of the Th differentiation states. 2

4 INTRODUCTION Complete deficiency of IL-2 has been implicated in severe combined immunodeficiency, whereas reduction of the IL-2 correlates with reduced function of CD4+CD25+ regulatory T cells and destabilization of immune homeostasis leading to autoimmune disease. Increased expression of IL-2 has also been implicated in inflammatory conditions such as inflammatory bowel disease and chronic liver diseases. IL-2 therefore is both a immune stimulator and immune suppressor cytokine which efficiently controls the immune system to deal with autoimmunity and adaptive immune response. IL-4 participates in at least several B-cell activation processes as well as of other cell types. It is a co-stimulator of DNA-synthesis. It induces the expression of class II MHC molecules on resting B-cells. It enhances both secretion and cell surface expression of IgE and IgG1. It also regulates the expression of the low affinity Fc receptor for IgE (CD23) on both lymphocytes and monocytes. IL-4 is secreted. 3

5 INTRODUCTION 2. ASSAY SUMMARY Remove appropriate number of antibody coated well strips. Equilibrate all reagents to room temperature. Prepare all reagents, samples, and standards as instructed. Add target specific standard or samples for both targets to appropriate wells (See Section 13). DO NOT add multiple target specific reagents to a single well. Add target specific Antibody Cocktail to appropriate wells. Incubate at room temperature. Aspirate and wash each well. Add TMB Substrate to each well and incubate. Add Stop Solution at a defined endpoint. Alternatively, record color development kinetically after TMB substrate addition. 4

6 GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY Store kit at 2-8ºC immediately upon receipt. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in sections 9, 10 & 11. 5

7 GENERAL INFORMATION 5. MATERIALS SUPPLIED 10X IL-2 Capture Antibody 300 µl Storage Condition (Before Preparation) +2-8ºC 10X IL-2 Detector Antibody 300 µl +2-8ºC 10X IL-4 Capture Antibody 300 µl +2-8ºC 10X IL-4 Detector Antibody 300 µl +2-8ºC IL-2 Human Lyophilized Recombinant Protein 1 Vial +2-8ºC IL-4 Human Lyophilized Recombinant Protein 1 Vial +2-8ºC Antibody Diluent 4BI 3 ml +2-8ºC Antibody Diluent CPI 3 ml +2-8ºC 10X Wash Buffer PT 20 ml +2-8ºC 5X Cell Extraction Buffer PTR* 10 ml +2-8ºC 50X Cell Extraction Enhancer Solution* 1 ml +2-8ºC TMB Substrate 12 ml +2-8ºC Stop Solution 12 ml +2-8ºC Sample Diluent NS 12 ml +2-8ºC Sample Diluent 25BS 12 ml +2-8ºC Sample Diluent 75BP SimpleStep Pre-Coated 96 Well Microplate (12 x 8 well strips) Plate Seal 12 ml +2-8ºC 96 Wells +2-8ºC ºC Item Amount * 5X Cell Extraction Buffer PTR and 50X Cell Extraction Enhancer Solution are only required for cell or tissue extract samples. 6

8 GENERAL INFORMATION 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: Microplate reader capable of measuring absorbance at 450 or 600 nm Method for determining protein concentration (BCA assay recommended) Deionized water PBS (1.4 mm KH2PO4, 8 mm Na2HPO4, 140 mm NaCl, 2.7 mm KCl, ph 7.4) Multi- and single-channel pipettes Tubes for standard dilution Plate shaker for all incubation steps Phenylmethylsulfonyl inhibitors) Fluoride (PMSF) (or other protease 7. LIMITATIONS Only 1 target can be measured per microplate well using this assay. DO NOT add multiple target specific reagents or samples to a single well Assay kit intended for research use only. Not for use in diagnostic procedures Do not use kit or components if it has exceeded the expiration date on the kit labels Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted 7

9 GENERAL INFORMATION 8. TECHNICAL HINTS Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers. Avoid foaming components Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions Ensure plates are properly sealed or covered during incubation steps Complete removal of all solutions and buffers during wash steps is necessary to minimize background As a guide, typical ranges of sample concentration for commonly used sample types are shown below in Sample Preparation (section 11) All samples should be mixed thoroughly and gently. Avoid multiple freeze/thaw of samples Incubate ELISA plates on a plate shaker during all incubation steps When generating positive control samples, it is advisable to change pipette tips after each step The provided 5X Cell Extraction Buffer contains phosphatase inhibitors. Protease inhibitors can be added if required The provided 50X Cell Extraction Enhancer Solution may precipitate when stored at + 4ºC. To dissolve, warm briefly at + 37ºC and mix gently. The 50X Cell Extraction Enhancer Solution can be stored at room temperature to avoid precipitation This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions or bubbles when mixing or reconstituting 8

10 ASSAY PREPARATION 9. REAGENT PREPARATION Equilibrate all reagents to room temperature (18-25 C) prior to use. The kit contains enough reagents for 96 wells. The sample volumes below are sufficient for 48 wells (6 x 8-well strips); adjust volumes as needed for the number of strips in your experiment. Prepare only as much reagent as is needed on the day of the experiment. Capture and Detector Antibodies have only been tested for stability in the provided 10X formulations X Wash Buffer PT Prepare 1X Wash Buffer PT by diluting 10X Wash Buffer PT with deionized water. To make 50 ml 1X Wash Buffer PT combine 5 ml 10X Wash Buffer PT with 45 ml deionized water. Mix thoroughly and gently. 9.2 IL-2 Antibody Cocktail Prepare IL-2 Antibody Cocktail by diluting the capture and detector antibodies in Antibody Diluent 4BI. To make 3 ml of the IL-2 Antibody Cocktail combine 300 µl 10X IL-2 Capture Antibody and 300 µl 10X IL-2 Detector Antibody with 2.4 ml Antibody Diluent 4BI. Mix thoroughly and gently. 9.3 IL-4 Antibody Cocktail Prepare IL-4 Antibody Cocktail by diluting in Antibody Diluent CPI. To make 3 ml of the IL-4 Antibody Cocktail combine 300 µl 10X Capture IL-4 Antibody and 300 µl 10X IL-4 Detector Antibody with 2.4 ml Antibody Diluent CPI. Mix thoroughly and gently X Cell Extraction Buffer PTR (For cell and tissue extracts only) If required, prepare 1X Cell Extraction Buffer PTR by diluting 5X Cell Extraction Buffer PTR and 50X Cell Extraction Enhancer Solution to 1X with deionized water. To make 10 ml 1X Cell Extraction Buffer PTR combine 7.8 ml 9

11 ASSAY PREPARATION deionized water, 2 ml 5X Cell Extraction Buffer PTR and 200 µl 50X Cell Extraction Enhancer Solution Mix thoroughly and gently. If required protease inhibitors can be added. Alternative Enhancer may be added to Cell Extraction Buffer after extraction of cells or tissue. Refer to note in Section

12 ASSAY PREPARATION 10. STANDARD PREPARATION - IL-2 Prepare serially diluted standards immediately prior to use. Always prepare a fresh set of positive controls for every use. The following table describes the preparation of a standard curve for duplicate measurements (recommended) Reconstitute the IL-2 Human Lyophilized Recombinant Protein standard sample by adding 100 µl water. Mix thoroughly and gently. Hold at room temperature for 1 minute. This is the 100 ng/ml Stock Standard Solution (see table below). Remaining stock material should be aliquoted and stored at -80 C Label eight tubes with # For serum/plasma samples measurements add 150 μl Sample Diluent 25BS to tubes #2-8. For cell culture supernatant samples measurements, add 150 μl Sample Diluent NS to tubes # Prepare a 2,000 pg/ml Standard #1 by adding 6 μl of the 100 ng/ml Stock Standard Solution to 294 µl of appropriate diluent to tube #1. Mix thoroughly and gently Prepare Standard #2 by transferring 150 μl from Standard #1 to tube #2. Mix thoroughly and gently Using the table below as a guide, repeat for Tubes #3 through # Standard #8 contains no protein and is the Blank control. Standard # Sample to Dilute Volume to Dilute (µl) (Blank) Stock Standard #1 Standard #2 Standard #3 Standard #4 Standard #5 Standard #6 none Volume of Diluent (µl) Starting Conc. (pg/ml) Final Conc. (pg/ml) 100,000 2,000 1, ,000 1,

13 ASSAY PREPARATION 11. STANDARD PREPARATION IL-4 Prepare serially diluted standards immediately prior to use. Always prepare a fresh set of positive controls for every use. The following table describes the preparation of a standard curve for duplicate measurements (recommended) For serum/plasma samples measurements, reconstitute the IL-4 Human Lyophilized Recombinant Protein standard sample by adding 200 µl Sample Diluent 75BP by pipette. For cell culture supernatant samples measurements, reconstitute the IL-4 Human Lyophilized Recombinant Protein standard sample by adding 200 µl water by pipette. If cell culture supernatants will be measured without dilution (see Section ), reconstitute the IL-4 Human Lyophilized Recombinant Protein standard sample by adding 200 µl your own cell culture media by pipette Mix thoroughly and gently. Hold at room temperature for 1 minute. This is the 2,000 pg/ml Stock Standard Solution (see table below). Remaining stock material should be aliquoted and stored at -80 C Label eight tubes with # Add μl appropriate diluent into tube # Prepare 1,000 pg/ml Standard #1 by adding μl of the 2,000 pg/ml Stock Standard Solution to µl of appropriate diluent to tube #1. Mix thoroughly and gently Prepare Standard #2 by transferring μl Standard #1 to tube #2. Mix thoroughly and gently. from Prepare Standard #3 by transferring μl Standard #2 to tube #3. Mix thoroughly and gently. from Using the table below as a guide, repeat for Tubes #4 through # Standard #8 contains no protein and is the Blank control. 12

14 ASSAY PREPARATION Standard # Sample to Dilute Volume to Dilute (µl) (Blank) Stock Standard #1 Standard #2 Standard #3 Standard #4 Standard #5 Standard #6 none 0 Volume of Diluent (µl) Starting Conc. (pg/ml) Final Conc. (pg/ml) 2,000 1, ,

15 ASSAY PREPARATION 12. SAMPLE PREPARATION TYPICAL SAMPLE DYNAMIC RANGE - IL-2 Sample Type Loading Dilution Range 48 hours PHA-stimulated PBMC supernatant % Normal Human Serum 1-50% Normal Human Plasma (Heparin) 1-50% Normal Human Plasma (EDTA) 1-50% TYPICAL SAMPLE DYNAMIC RANGE - IL-4 Sample Type 48 hours PHA-stimulated PBMC supernatant Range 11X diluted Neat 12.1 Plasma IL-2 Plasma Preparation Plasma samples must be diluted 2X with Sample Diluent NS prior to loading on the plate to ensure good linearity and recoverability of the protein. If greater dilution is desired, the Sample Diluent 25BS must be diluted by an equivalent factor in Sample Diluent NS before being used to dilute standards. Store un-diluted plasma samples at -20ºC or below for up to 3 months. Avoid repeated freeze-thaw cycles IL-4 Plasma Preparation Collect plasma using citrate, EDTA or heparin. Centrifuge samples at 2,000 x g for 10 minutes. Neat plasma samples can be assayed without dilution. If needed, dilute samples into Sample 14

16 ASSAY PREPARATION Diluent 75BP and assay. Store un-diluted plasma samples at -20ºC or below for up to 3 months. Avoid repeated freeze-thaw cycles Serum IL-2 Serum Preparation Serum samples must be diluted 2X with Sample Diluent NS prior to loading on the plate to ensure good linearity and recoverability of the protein. If greater dilution is desired, the Sample Diluent 25BS must be diluted by an equivalent factor in Sample Diluent NS before being used to dilute standards. Store un-diluted serum at -20ºC or below. Avoid repeated freeze-thaw cycles IL-4 Serum Preparation Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2,000 x g for 10 minutes and collect serum. Neat serum samples can be assayed without dilution. If needed, dilute samples into Sample Diluent 75BP and assay. Store un-diluted serum at -20ºC or below. Avoid repeated freeze-thaw cycles Cell culture supernatant samples IL-2 Cell culture supernatants Centrifuge cell culture media at 2,000 x g for 10 minutes to remove debris. Cell culture supernatants should be diluted at least ten fold in Sample Diluent NS to prevent saturation of the signal. Store samples at -20 C or below. Avoid repeated freezethaw cycles IL-4 Cell culture supernatants Centrifuge cell culture media at 2,000 x g for 10 minutes to remove debris. Collect supernatants, dilute them at least two fold in Sample Diluent NS and assay. If low concentrations of IL-4 are 15

17 ASSAY PREPARATION expected, it is preferable to assay the samples without dilution. Store samples at -20 C or below. Avoid repeated freeze-thaw cycles. 16

18 ASSAY PREPARATION 13. PLATE PREPARATION The 96 well plate strips included with this kit are supplied ready to use. It is not necessary to rinse the plate prior to adding reagents Unused plate strips should be immediately returned to the foil pouch containing the desiccant pack, resealed and stored at 4 C For each assay performed, a minimum of two wells must be used as the zero control For statistical reasons, we recommend each sample should be assayed with a minimum of two replicates (duplicates) Differences in well absorbance or edge effects have not been observed with this assay We recommend following the layout shown below: 17

19 ASSAY PROCEDURE 14. ASSAY PROCEDURE Equilibrate all materials and prepared reagents to room temperature prior to use. It is recommended to assay all standards, controls and samples in duplicate Prepare all reagents, working standards, and samples as directed in the previous sections Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, reseal and return to 4ºC storage Add 50 µl of each sample or target specific standards to appropriate wells Add 50 µl of IL-2 or IL-4 Antibody Cocktail to each well used for the respective target Seal the plate and incubate for 1 hour at room temperature on a plate shaker set to 400 rpm Wash each well with 3 x 350 µl 1X Wash Buffer PT. Wash by aspirating or decanting from wells then dispensing 350 µl 1X Wash Buffer PT into each well. Complete removal of liquid at each step is essential for good performance. After the last wash invert the plate and blot it against clean paper towels to remove excess liquid Add 100 µl of TMB Substrate to each well and incubate for 10 minutes in the dark on a plate shaker set to 400 rpm Add 100 µl of Stop Solution to each well. Shake plate on a plate shaker for 1 minute to mix. Record the OD at 450 nm. This is an endpoint reading. Alternative to : Instead of the endpoint reading at 450 nm, record the development of TMB Substrate kinetically. Immediately after addition of TMB Development Solution begin recording the blue color development with elapsed time in the microplate reader prepared with the following settings: 18

20 ASSAY PROCEDURE Mode: Kinetic Wavelength: 600 nm Time: up to 15 min Interval: 20 sec - 1 min Shaking: Shake between readings Note that an endpoint reading can also be recorded at the completion of the kinetic read by adding 100 µl Stop Solution to each well and recording the OD at 450 nm Analyze the data as described below. 19

21 DATA ANALYSIS 15. CALCULATIONS Subtract average zero standard from all readings. Average the duplicate readings of the positive control dilutions and plot against their concentrations. Draw the best smooth curve through these points to construct a standard curve. Most plate reader software or graphing software can plot these values and curve fit. A four parameter algorithm (4PL) usually provides the best fit, though other equations can be examined to see which provides the most accurate (e.g. linear, semi-log, log/log, 4 parameter logistic). Interpolate protein concentrations for unknown samples from the standard curve plotted. Samples producing signals greater than that of the highest standard should be further diluted and reanalyzed, then multiplying the concentration found by the appropriate dilution factor. 20

22 DATA ANALYSIS 16. TYPICAL DATA IL-2 TYPICAL IL-2 STANDARD CURVE Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed. Standard Curve Measurements with Sample Diluent NS Conc. O.D. 450 nm (pg/ml) 1 2 Mean O.D , , Figure 1. Example IL-2 standard curve in sample diluents NS. The IL-2 standard curve was prepared as described in Section 10. Raw data values are shown in the table. Background-subtracted data values (mean +/- SD) are graphed. 21

23 DATA ANALYSIS Standard Curve Measurements with Standard Diluent 25BS Conc. O.D. 450 nm Mean (pg/ml) 1 2 O.D , , Figure 2. Example IL-2 standard curve in standard diluent 25BS. The curve was prepared by loading standard curve as described in Section 10. Raw data values are shown in the table. Background-subtracted data values (mean +/- SD) are graphed. 22

24 DATA ANALYSIS IL-2 SENSITIVITY The calculated minimal detectable (MDD) dose is 9 pg/ml. The MDD was determined by calculating the mean of zero standard replicates (n=24) and adding 2 standard deviations then extrapolating the corresponding concentrations. Ten individual healthy donors were evaluated for the presence of IL-2 in serum using this assay. All samples measured less than the lowest IL-2 standard of pg/ml. IL-2 RECOVERY (Sample spiking in representative sample matrices) Sample Type 10% Culture Media 50% Normal Human Serum 50% Normal Human Plasma (Sodium Citrate) 50% Normal Human Plasma (Heparin) 50% Normal Human Plasma (EDTA) Average % Recovery Range (%) IL-2 LINEARITY OF DILUTION NHS 50% NHP Citrate 50% NHP EDTA 50% NHP Heparin 50% Media 10% 1:1 Interpolated value (pg/ml) :2 % Expected value Interpolated value (pg/ml) :4 % Expected value Interpolated value (pg/ml) :8 % Expected value Interpolated value (pg/ml) % Expected value

25 DATA ANALYSIS IL-2 PRECISION Mean coefficient of variations of interpolated values from 2 concentrations of IL-2 protein within the working range of the assay. IntraAssay 6 6% n= CV (%) InterAssay 24 6% IL-2 SAMPLE VALUES IL-2 (pg/ml) PHA + PHA - Figure 3. Specificity of IL-2 signal on stimulated and non stimulated media supernatants. Human PBMCs were cultured in RPMI supplemented with 10% fetal calf serum, 2 mm L-glutamine, 100 U/mL penicillin, and 100 µg/ml streptomycin. Cells were cultured for 2 days at 37⁰C in the presence or absence of PHA. The concentrations of IL-2 were interpolated from the calibration curve and corrected for sample dilution. The mean IL-2 concentration was determined to be 16 pg/ml in unstimulated PBMC supernatants and 11,460 pg/ml in stimulated PBMC supernatants. 24

26 DATA ANALYSIS 17. TYPICAL DATA IL-4 TYPICAL IL-4 STANDARD CURVE Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed. Standard Curve Measurements Conc. O.D. 450 nm Mean (pg/ml) 1 2 O.D , Figure 4. Example of IL-4 standard curve using Sample Diluent NS for cell culture supernatant samples measurements. The IL-4 standard curve was prepared as described in Section 11. Raw data values are shown in the table. Background-subtracted data values (mean +/- SD) are graphed. 25

27 DATA ANALYSIS Standard Curve Measurements Conc. O.D. 450 nm Mean (pg/ml) 1 2 O.D , Figure 5. Example of IL-4 standard curve using cell culture media for cell culture supernatant samples measurements. The IL-4 standard curve was prepared as described in Section 11. Raw data values are shown in the table. Background-subtracted data values (mean +/- SD) are graphed. 26

28 DATA ANALYSIS IL-4 SENSITIVITY The calculated minimal detectable (MDD) dose is equal to or less than 2 pg/ml. The MDD was determined by calculating the mean of zero standard replicates (n=30) and adding 2 standard deviations then extrapolating the corresponding concentrations. IL-4 RECOVERY (Sample spiking in representative sample matrices) Average % Recovery Sample Type 50% media (10F RPMI) Neat normal Human serum Neat normal Human plasma-edta Neat normal Human plasma-citrate Neat normal Human plasma-heparin Range (%) IL-4 LINEARITY OF DILUTION Dilution Factor Undiluted Dilution Factor Undiluted PHA PBMC SN (fold of dilution) Undiluted Interpolated value (pg/ml) % Media (10F RPMI) + IL-4 (pg/ml) Interpolated value (pg/ml) % Expected Value % Expected Value

29 DATA ANALYSIS Dilution Factor Undiluted Dilution Factor Undiluted Dilution Factor Undiluted Dilution Factor Undiluted % Media (10F RPMI) + IL-4 (pg/ml) Interpolated value (pg/ml) Neat Human serum + IL-4 (pg/ml) Interpolated value (pg/ml) Neat Human plasma-edta + IL-4 (pg/ml) Interpolated value (pg/ml) Neat Human plasma-citrate + IL-4 (pg/ml) Interpolated value (pg/ml) % Expected Value % Expected Value % Expected Value % Expected Value

30 DATA ANALYSIS Dilution Factor Undiluted Neat Human plasma-heparin + IL-4 (pg/ml) Interpolated value (pg/ml) % Expected Value IL-4 PRECISION Mean coefficient of variations of interpolated values from 3 concentrations of PHA-stimulated PBMC supernatant within the working range of the assay. n= CV (%) IntraAssay InterAssay

31 DATA ANALYSIS 18. SPECIES REACTIVITY This kit detects IL-2 in Human plasma and serum samples and IL-4 in Human cell culture supernatant, plasma and serum samples. It does not cross-react with mouse IL-2 protein at 10 ng/ml Please contact our Technical Support team for more information. 30

32 RESOURCES 19. TROUBLESHOOTING Problem Cause Solution Difficulty pipetting lysate; viscous lysate. Genomic DNA solubilized Prepare 1X Cell Extraction Buffer PTR (without enhancer). Add enhancer to lysate after extraction. Inaccurate Pipetting Check pipettes Improper standard dilution Prior to opening, briefly spin the stock standard tube and dissolve the powder thoroughly by gentle mixing Incubation times too brief Ensure sufficient incubation times; increase to 2 or 3 hour standard/sample incubation Inadequate reagent volumes or improper dilution Check pipettes and ensure correct preparation Incubation times with TMB too brief Ensure sufficient incubation time until blue color develops prior addition of Stop solution Plate is insufficiently washed Review manual for proper wash technique. If using a plate washer, check all ports for obstructions. Contaminated wash buffer Prepare fresh wash buffer Improper storage of the ELISA kit Store your reconstituted standards at -80 C, all other assay components 4 C. Keep TMB substrate solution protected from light. Poor standard curve Low Signal Large CV Low sensitivity 31

33 RESOURCES 20. NOTES 32

34 RESOURCES 33

35 RESOURCES 34

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