ab IL-1a (Interleukin-1a) + IL-6 (Interleukin-6) Human SimpleStep ELISA Kit

Transcription

1 ab IL-1a (Interleukin-1a) + IL-6 (Interleukin-6) Human SimpleStep ELISA Kit Instructions for Use For the simultaneous quantitative measurement of IL-1a in parallel with IL-6 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 5 February 2014

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-1a STANDARD PREPARATION IL-6 SAMPLE PREPARATION PLATE PREPARATION ASSAY PROCEDURE 14. ASSAY PROCEDURE 18 DATA ANALYSIS CALCULATIONS TYPICAL DATA IL-1a TYPICAL DATA IL-6 SPECIES REACTIVITY RESOURCES TROUBLESHOOTING NOTES

3 INTRODUCTION 1. BACKGROUND Abcam s IL-1a (Interleukin-1 alpha) and IL-6 (Interleukin-6) in vitro multi-target SimpleStep ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the simultaneous, accurate and quantitative measurement of IL-1a in parallel with IL-6 in Human cell culture supernatant, plasma and serum samples. The multi-target SimpleStep ELISA measures a single target per well. For individual targets an affinity tag labeled capture antibody and a reporter conjugated detector antibody immunocapture the sample analyte in solution. Within each individual target well, this entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the individual targets antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change 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. Interleukin 1 (IL-1) refers to two proteins encoded by two different genes (IL-1a and IL-1b), both of which share the same cell surface receptors. IL-1a is a proinflammatory cytokine typically expressed under normal conditions in skin keratinocytes, some epithelial cells as well as some cells in the central nervous system. Under pathological conditions, IL-1a is also strongly expressed by monocytes, tissue macrophages, dendritic cells, B lymphocytes and NK cells. IL-1a is synthesized as a 31kDa precursor, some of which is cleaved prior to secretion into a 17.5kDa protein. IL-1a is biologically active both in a paracrine or systemic mode of action. The paracrine effects are mediated by the uncleaved portion of IL-1a, which is transported to the cell surface where it activates the IL-1 receptor type I (IL-1RI) of 2

4 INTRODUCTION adjacent cells. The systemic effect is mediated by cleaved IL-1a, which is secreted into the bloodstream. Interleukin 6 (IL-6) is a cytokine with a wide variety of biological functions. It is a potent inducer of the acute phase response and plays an essential role in the final differentiation of B-cells into Ig-secreting cells. IL-6 is involved in lymphocyte and monocyte differentiation and IL-6 induces myeloma and plasmacytoma growth as well as nerve cells differentiation. B-cells, T-cells, hepatocytes, hematopoeitic progenitor cells and cells of the CNS are all responsive to IL-6. IL-6 is discharged into the bloodstream after muscle contraction and acts to increase the breakdown of fats and to improve insulin resistance 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-1a Capture Antibody (Lyophilized) 300 µl Storage Condition (Before Preparation) +2-8ºC 10X IL-1a Detector Antibody 300 µl +2-8ºC 10X IL-6 Capture Antibody 300 µl +2-8ºC 20X IL-6 Detector Antibody µl +2-8ºC IL-1a Human Lyophilized Recombinant Protein 1 Vial +2-8ºC IL-6 Human Lyophilized Recombinant Protein 1 Vial +2-8ºC Antibody Diluent CPI 2 x 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 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 The provided Antibody Diluent CPI and Sample Diluent NS contain protease inhibitor aprotinin. Additional protease inhibitors can be added if required or bubbles when mixing or reconstituting 8

10 GENERAL INFORMATION 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 9

11 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 X IL-1a Capture Antibody Prepare the 20X IL-1a Capture Antibody by reconstituting the lyophilized IL-1a Capture Antibody in nanopure water. To reconstitute the IL-1a Capture Antibody, centrifuge the vial at 10,000 x g for 2 minutes and then add µl of nanopure water per vial; incubate at room temperature for 5 minutes and vortex well ensuring the material is fully resuspended. For long term storage, the reconstituted 20X IL-1a Capture Antibody should be aliquoted and frozen at -20 C. Avoid repeat freeze/thaw cycles 9.3 IL-1a Antibody Cocktail Prepare IL-1a Antibody Cocktail by diluting in the 20X IL-1a Capture Antibody and 10X IL-1a Detector Antibody in Antibody Diluent CPI. To make 3 ml of the IL-1a Antibody Cocktail combine µl 20X IL-1a Capture Antibody and 300 µl 10X IL-1a Detector Antibody with 2.55 ml Antibody Diluent CPI. Mix thoroughly and gently. 10

12 ASSAY PREPARATION 9.4 IL-6 Antibody Cocktail Prepare IL-6 Antibody Cocktail by diluting in Antibody Diluent CPI. To make 3 ml of the IL-6 Antibody Cocktail combine 300 µl 10X Capture IL-6 Antibody and µl 20X IL-6 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 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

13 ASSAY PREPARATION 10. STANDARD PREPARATION - IL-1a 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 Human Lyophilized IL-1a standard sample by adding 1,000 µl of Sample Diluent NS by pipette. Mix thoroughly and gently. Hold at room temperature for 3 minutes and mix gently. This is the 10,000 pg/ml Stock Standard Solution (see table below) Label eight tubes with numbers For measuring serum or plasma samples, add μl of Sample Diluent 25BS into tube numbers For measuring cell culture media samples, add μl of non-reactive base media Prepare 200 pg/ml Standard #1 by adding 7 μl of the 10,000 pg/ml Stock Standard Solution to 343 µl of appropriate diluent. Mix thoroughly and gently Prepare Standard #2 by transferring μl Standard #1 to tube #2. Mix thoroughly and gently. from 10.6 Prepare Standard #3 by transferring μl Standard #2 to tube #3. Mix thoroughly and gently. from 10.7 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 IL-1a Standard # Sample to Dilute Volume to Dilute (µl) (Blank) Stock Standard #1 Standard #2 Standard #3 Standard #4 Standard #5 Standard #6 none 7 - Volume of Diluent (µl) 343 Starting Conc. (pg/ml) Final Conc. (pg/ml) 10,

15 ASSAY PREPARATION 11. STANDARD PREPARATION IL-6 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-6 standard sample by adding 500 µl Sample Diluent NS by pipette. Mix thoroughly and gently. Hold at room temperature for 3 minutes and mix gently. This is the 5,000 pg/ml Stock Standard Solution (see table below) Label eight tubes with numbers Add μl Sample Diluent NS into tube numbers Prepare 1,000 pg/ml Standard #1 by adding 50 μl of the 5 ng/ml Stock Standard Solution to 200 µl of Sample Diluent NS 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. IL-6 Standard # Sample to Dilute Volume to Dilute (µl) (Blank) Stock Standard #1 Standard #2 Standard #3 Standard #4 Standard #5 Standard #6 none 50 0 Volume of Diluent (µl) 200 Starting Conc. (pg/ml) Final Conc. (pg/ml) 5,000 1, ,

16 ASSAY PREPARATION 12. SAMPLE PREPARATION TYPICAL SAMPLE DYNAMIC RANGE - IL-1a Sample Type Range Human Serum Human Plasma Citrate Human Plasma Heparin Human Plasma EDTA 1 25 Conditioned Media TYPICAL SAMPLE DYNAMIC RANGE - IL-6 Sample Type Range PBMC (PHA stimulated) tissue culture supernatant 1:25 1:1,000 dilution 12.1 Plasma IL-1a Plasma Preparation Collect plasma using citrate, EDTA or heparin. Centrifuge samples at 2,000 x g for 10 minutes. The assay plate may be loaded with neat citrate and heparin plasma however, if desired, samples may be diluted into Sample Diluent NS to achieve maximal recovery. EDTA plasma must be diluted at least 1:4 in Sample Diluent NS to achieve maximal recovery. Store un-diluted plasma samples at -20ºC or below for up to 3 months. Avoid repeated freeze-thaw cycles IL-6 Plasma Preparation Collect plasma using citrate, EDTA or heparin. Centrifuge samples at 2,000 x g for 10 minutes. Dilute samples into Sample Diluent NS and assay. 15

17 ASSAY PREPARATION Store un-diluted plasma samples at -20ºC or below for up to 3 months. Avoid repeated freeze-thaw cycles Serum IL-1a 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. The assay plate may be loaded with neat serum however, if it is necessary, samples may be diluted into Sample Diluent 25BS to achieve maximal recovery. Store un-diluted serum at -20ºC or below. Avoid repeated freeze-thaw cycles IL-6 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. Dilute samples into Sample Diluent NS and assay. Store un-diluted serum at -20ºC or below. Avoid repeated freeze-thaw cycles Cell culture supernatant samples (IL-1a & IL-8) Centrifuge cell culture media at 2,000 x g for 10 minutes to remove debris. Cell culture supernatants can be used without dilution. If required, cell culture supernatant samples should be diluted in Sample Diluent NS. 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-1a or IL-6 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-1a TYPICAL IL-1a 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 of IL-1a standard curve in sample diluent NS. The IL-1a standard curve was prepared as described in Section 10 using Sample Diluent NS. Raw data values are shown in the table. Background-subtracted data values (mean +/- SD) are graphed. 21

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

24 DATA ANALYSIS Standard Curve Measurements Conc. O.D. 450 nm Mean (pg/ml) 1 2 O.D Figure 3. Example of IL-1a standard curve in non reactive culture media. The IL-1a standard curve was prepared as described in Section 10 using non reactive culture media. Raw data values are shown in the table. Backgroundsubtracted data values (mean +/- SD) are graphed. 23

25 DATA ANALYSIS IL-1a SENSITIVITY The calculated minimal detectable (MDD) dose is 0.5 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. IL-1a RECOVERY Three concentrations of IL-1a were spiked in duplicate to the indicated biological matrix to evaluate signal recovery in the working range of the assay. Sample Type 100% Cell Culture Media 100% Human Serum 100% Human Plasma-Citrate 100% Human Plasma-Heparin 25% Human Plasma-EDTA Average % Recovery Range (%)

26 DATA ANALYSIS IL-1a LINEARITY OF DILUTION Linearity of dilution is determined based on interpolated values from the standard curve. Linearity of dilution defines a sample concentration interval in which interpolated target concentrations are directly proportional to sample dilution. 1:1 1:2 1:4 1:8 Interpolated value (pg/ml) % Expected value Interpolated value (pg/ml) % Expected value Interpolated value (pg/ml) % Expected value Interpolated value (pg/ml) % Expected value NHS 100% NHP Citrate 100% NHP Heparin 100% NHP EDTA 25% Media 100% IL-1a PRECISION Mean coefficient of variations of interpolated values from 3 concentrations of HeLa extracts within the working range of the assay. n= CV (%) IntraAssay InterAssay

27 DATA ANALYSIS IL-1a ASSAY SPECIFICITY Ten individual healthy donors were evaluated for the presence of IL-1a in serum using this assay. All samples measure less than the lowest IL1-a standard of 1.6 pg/ml Figure 4. Specificity of IL1-a signal on stimulated and non stimulated media supernatants. Human PBMC 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 37ºC in the presence or absence of PHA. The concentrations of IL-1a were interpolated from the calibration curve and corrected for sample dilution. The mean IL-1a concentration was determined to be 1.4 pg/ml in unstimulated PBMC supernatants and 96 pg/ml in stimulated PBMCs 26

28 DATA ANALYSIS 17. TYPICAL DATA IL-6 TYPICAL IL-6 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 5. Example of IL-6 standard curve. The IL-6 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. 27

29 DATA ANALYSIS IL-6 SENSITIVITY The calculated minimal detectable (MDD) dose is 16 pg/ml. The MDD was determined by calculating the mean of zero standard replicates (n=18) and adding 2 standard deviations then extrapolating the corresponding concentrations. IL-6 RECOVERY Three concentrations of IL-6 were spiked in duplicate to the indicated biological matrix to evaluate signal recovery in the working range of the assay. Sample Type 100% Cell Culture Media 10% Normal Human Serum 10% Normal Human Plasma - EDTA 10% Normal Human Plasma - Citrate 10% Normal Human Plasma - Heparin Average % Recovery Range (%)

30 DATA ANALYSIS IL-6 LINEARITY OF DILUTION Linearity of dilution is determined based on interpolated values from the standard curve. Linearity of dilution defines a sample concentration interval in which interpolated target concentrations are directly proportional to sample dilution. Dilution Factor Neat Interpolated value (pg/ml) % Expected value Interpolated value (pg/ml) % Expected value Interpolated value (pg/ml) % Expected value Interpolated value (pg/ml) % Expected value Interpolated value (pg/ml) % Expected value Media 50% 10% Serum

31 DATA ANALYSIS Dilution Factor Neat Interpolated value (pg/ml) % Expected value Interpolated value (pg/ml) % Expected value Interpolated value (pg/ml) % Expected value Interpolated value (pg/ml) % Expected value Interpolated value (pg/ml) % Expected value 10% Plasma (EDTA) 10% Plasma (Heparin) 10% Plasma (Citrate) IL-6 PRECISION Mean coefficient of variations of interpolated values from 2 concentrations of PHA stimulated PBMC cell culture supernatant within the working range of the assay. n= CV (%) IntraAssay InterAssay

32 DATA ANALYSIS IL-6 ASSAY SPECIFICITY Serum/Plasma. Ten serum samples from apparently healthy donors were diluted 1:10 in Sample Diluent NS and tested for IL-6 using this kit. OD 450nm values for all donors were less than the lowest tested concentration of the standard curve (16 pg/ml). Cell Culture Supernatants. Human peripheral blood mononuclear cells (PBMCs) were cultured in the absence or presence of phytohemagglutinin (PHA) for 2 days. Cell culture supernatants were assayed using this kit for levels of IL-6. Results as follows: Condition Unstimulated PHA Day 2 (pg/ml) 1,027 16,670 Figure 6. Comparison of secreted IL-6 in unstimulated and PHA-stimulated Human PBMC. PBMC were grown in the absence or presence of phytohemagglutinin (PHA) for 2 days. IL-6 was measured in 100, 200, 400 and 800-fold diluted cell culture supernatants of unstimulated and PHA stimulated PBMC. Raw data values (mean +/-SD, n=2) are graphed. 31

33 DATA ANALYSIS 18. SPECIES REACTIVITY This kit detects IL-1a in parallel with IL-6 in Human cell culture supernatant, plasma and serum samples. Please contact our Technical Support team for more information. 32

34 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 Low sensitivity 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. Precipitate in Diluent Precipitation and/or coagulation of components within the Diluent. Precipitate can be removed by gently warming the Diluent to 37ºC. Poor standard curve Low Signal Large CV 33

35 RESOURCES 20. NOTES 34

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