INSTRUCTIONS ImmuLux Human IL- Fluorescent ELISA Kit 3747 N. Meridian Road P.O. Box 7 Rockford, IL 05 840 53w Product Description Number Description 840 ImmuLux Human IL- Fluorescent ELISA Kit The Pierce Endogen ImmuLux Human IL- Fluorescent ELISA is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human Interleukin- in serum; EDTA, heparin and sodium citrate plasma; and culture supernatants. For Research Use Only. Not for use in diagnostic procedures. Introduction The ImmuLux Human IL- Kit is a sandwich ELISA. The microplate provided was pre-coated with an antibody specific to IL-. This antibody captures any IL- in the standards and samples added to the plate. After unbound proteins are washed away, the biotinylated detecting antibody is added and binds to a second site on the IL- protein. After washing away excess detecting antibody, streptavidin-horseradish peroxidase (SA-HRP) is added. HRP is an enzyme that reacts with a variety of substrates to produce signal. QuantaBlu Fluorogenic Peroxidase Substrate (U.S. Patent #,040,50 and other patents pending) is used in this assay. The enzyme-substrate reaction generates a fluorescent signal that is read on a fluorometric plate reader. The amount of signal produced is proportional to the amount of IL- in the original standard or sample. Materials Provided Anti-Human IL- Pre-coated 9-well Plate, Lyophilized Recombinant Human IL- Standard, Standard Diluent, 5 ml, contains 0.% sodium azide Biotinylated Antibody Reagent, ml, contains 0.% sodium azide Streptavidin-HRP Reagent, ml Stable Peroxide Solution,.5 ml QuantaBlu Substrate Solution, 3 ml Stop Solution, 3 ml Wash Buffer, 50 ml Plate Sealers, 3 Storage Instructions Store all reagents at -8ºC upon receipt. Do not freeze reagents. Do not use after expiration date on kit box.
Summary of Procedure Step Add 50 µl of Sample Diluent to each well being used. Add 50 µl of Standards and samples, in duplicate. Step Incubate the covered plate at room temperature, 0-5 C, for hour. Step 3 Wash the plate THREE times. Step 4 Add 00 µl of biotinylated antibody reagent to each well. Incubate the covered plate at room temperature, 0-5 C, for hour. Step 5 Wash the plate THREE times. Step Add 00 µl of Streptavidin-HRP to each well. Step 7 Incubate the covered plate at room temperature, 0-5 C, for 30 minutes. Step 9 Prepare the QuantaBlu Substrate. Add 00 µl to each well. Step 8 Wash the plate THREE times. Step 0 Incubate the covered plate at room temperature, 0-5 C, for 30 minutes. Step Stop reaction by adding 00 µl of Stop Solution to each well. Step Read fluorescence on a plate reader set at 35 nm excitation and 40 nm emission.
Additional Materials Required Fluorescent plate reader for 9-well plates Distilled or deionized water Precision pipettes with disposable plastic tips to deliver 5-,000 µl Plastic pipettes to deliver 5-5 ml Disposable reservoirs, if a multichannel pipette will be used A glass or plastic liter container for preparation of Wash Buffer A squirt wash bottle or automated plate washer.5 ml polypropylene or polyethylene tubes for preparation of standards (Do not use polystyrene, polycarbonate or glass tubes) 5 ml plastic tube for substrate preparation Precautions Review the Instruction Booklet carefully and verify all components against the materials provided list (page ) before beginning the assay. Bring all specimens and reagents to room temperature (0-5ºC) before use. Thaw samples and reagents at room temperature. Do not use heated water baths. Do not mix reagents from different kit lots. Do not use kit beyond its expiration date. Use fresh disposable pipette tips for each transfer to avoid cross-contamination. Use a new disposable reservoir and plate sealer for each step in the ELISA. Avoid exposure of reagents to excessive heat or light during storage and incubation. Individual components of this assay kit contain preservatives and antibiotics. Wear gloves while performing the assay to avoid contact with samples and reagents. Sample Handling Samples that are to be assayed within 4 hours may be stored at -8ºC. Samples that are to be stored for longer periods of time should be frozen in aliquots at -70ºC. Avoid multiple freeze-thaws. Bring samples to room temperature before running the assay. Mix samples by gently inverting the tubes. Do not use heated water baths to thaw samples. If using samples that are clotted, grossly hemolized or microbially contaminated, or if there is any question about the integrity of a sample, make a note on the template and interpret results with caution. If testing culture supernatants and using a medium other than RPMI with 5-0% FCS, we recommend that you validate the medium. To perform this validation, prepare two standard curves, one using your culture medium and the other using Standard Diluent. If the mean values of the Relative Fluorescent Units (RFU) for each point on the two curves are within 0% of each other, the medium will not interfere with the assay. If the RFU of the two curves differ by more than 0%, contact Technical Assistance for more information. 3
Sample Dilution If you suspect that the concentration of IL- in a sample exceeds the highest point on the standard curve; i.e., 4,000 pg/ml, prepare one or more five-fold dilutions of the test sample. A five-fold dilution is prepared by adding 50 µl of test sample to 00 µl of appropriate diluent. When testing culture supernatants, prepare the serial dilutions using your culture medium. When testing serum or plasma, prepare the serial dilutions using the Standard Diluent provided. Reagent Preparation Wash Buffer Label a clean glass or plastic bottle Wash Buffer. Add the entire contents of the 30X Wash Buffer bottle and dilute with distilled or deionized water to a final volume of.5 liters. Mix thoroughly. Wash Buffer should be stored at room temperature. Standards. Two vials of lyophilized standard are provided with this kit. Reconstitute and use one vial per assay. Use standards within one hour of reconstitution.. Reconstitute standard in distilled or deionized water. Reconstitution volume is stated on the standard vial label. The standard will take approximately minute to dissolve. Mix by gently inverting the vial. 3. Label eight tubes, one for each standard curve point: 4,000;,000; 50; 3; ; 4; and 0 pg/ml. 4. Pipette 300 µl of appropriate diluent into each tube. If testing serum, plasma or culture supernatants generated with RPMI containing 5-0% FCS, use the Standard Diluent provided to prepare standard dilutions. If testing culture supernatants generated with other types of media or media containing additives, we recommend that the media be validated in order to confirm that it will not interfere with the assay. To perform this validation, prepare two standard curves, one using your culture medium and the other using Standard Diluent. If the mean values of the Relative Fluorescent Units (RFU) for each point on the two curves are within 0% of each other, the medium will not interfere with the assay. The culture samples may be evaluated using a standard curve prepared with Standard Diluent. If the RFU of the two curves differ by more than 0%, contact Technical Assistance for more information. 5. Dilute the reconstituted standard : by adding 300 µl of the reconstituted standard to the tube labeled 4,000 pg/ml, mix gently. Prepare :4 serial dilutions as follows: Pipette 00 µl of the 4,000 pg/ml standard into the tube labeled,000 pg/ml and mix. Pipette 00 µl of the,000 pg/ml standard into the tube labeled 50 pg/ml and mix. Perform four additional serial dilutions. Do not add Standard to the tube marked 0 pg/ml. 4
Assay Procedure Note: Follow the Wash Procedure carefully. Remember to use a fresh pipette tip for each transfer. If using a multichannel pipette, use a new disposable reservoir for each assay reagent. All incubations should be performed on the benchtop without shaking.. Use the Data Template provided to record the locations of the Human IL- Standards and test samples. Seven Standards and one zero Standard should be run in duplicate with each series of unknown samples. If running a half plate, remove strips from the plate frame and store them in the foil pouch with desiccant provided for future use. Remember to save the plate frame at the end of the assay.. Add 50 µl of Standard Diluent to each well being used. Add 50 µl of Standard or sample to each well. Standards and samples should be run in duplicate. Cover plate with adhesive plate sealer. Incubate for hour at room temperature, 0-5ºC. 3. Thoroughly wash the plate THREE times with prepared Wash Buffer and pat dry. Wash Procedure Gently squeeze the sides of the plate frame when washing the plate to assure that all strips remain securely in the frame. Manual Wash: Decant the contents of the plate into a sink or other receptacle. Using a squirt bottle, vigorously fill each well completely with Wash Buffer, then decant the contents into a sink or other receptacle. Repeat the procedure two more times for a total of THREE washes. Pat plate onto paper towels or other absorbent material. Automated Wash: Aspirate all wells. Fill each well completely with wash buffer and aspirate. Repeat the procedure two more times for a total of THREE washes. Observe washing to ensure that all wells are being filled uniformly. 4. Add 00 µl of Biotinylated Antibody Reagent to each well being utilized. Cover plate with adhesive plate sealer. Incubate for hour at room temperature, 0-5ºC. 5. Thoroughly wash the plate THREE times with prepared Wash Buffer and pat dry.. Add 00 µl of Streptavidin-HRP Reagent to each well being utilized. Cover plate with adhesive plate sealer. Incubate for 30 minutes at room temperature, 0-5ºC. 7. Thoroughly wash the plate THREE times with prepared Wash Buffer and pat dry. 8. Prepare QuantaBlu Substrate by adding. ml of Stable Peroxide to 0.8 ml of QuantaBlu Substrate Solution. (Add 0. ml of Stable Peroxide to 5.4 ml of QuantaBlu Substrate Solution if running a half plate.) Mix gently and add 00 µl to each well. Incubate for 30 minutes at room temperature, 0-5ºC. 9. Add 00 µl of Stop Solution to each well. 0. Read the fluorescence at an excitation wavelength of 35 nm and an emission wavelength of 40 nm. Select the gain setting that generates the best signal-to-noise ratio. (Strong readings for the first seven standard points with a low value for the zero.) The plate must be read within 30 minutes of stopping the reaction. Instrumentation Pierce Endogen ImmuLux ELISA Kits were developed and tested using the CytoFluor 4000 Fluorescence Multi-Well Plate Reader from PerSeptive Biosystems, Inc. A gain setting of 90 is recommended for this instrument. Optimal gain settings and Relative Fluorescence Units will vary from one instrument to another. Because a standard curve is run with each assay, calculated sample values should be consistent from one instrument to another. Calculation of Results The standard curve is generated by plotting the average relative fluorescent units (RFU) obtained for each of the Standards on the vertical (Y) axis vs. the corresponding IL- concentrations on the horizontal (X) axis. If using a curve-fitting software package, plot a cubic spline or point-to-point curve fit. The amount of IL- in each sample is determined by interpolating from the fluorescent units to the IL- concentration using the standard curve. If a dilution was performed on a test sample, multiply the value interpolated from the standard curve by the dilution factor to calculate the pg/ml of IL- in the sample. 5
Human IL- Standard Curve Relative Fluorescence Units 70,000 0,000 50,000 40,000 30,000 0,000 0,000 0 0,000,000 3,000 4,000 Human IL- Performance Characteristics Standard Curve Range: to 4,000 pg/ml Suggested Standard Curve Points: 4,000,,000, 50, 3,, 4,, 0 Alternate curve points may be selected, but should not exceed the highest point of the standard curve, 4,000 pg/ml, or the lower limit of detection, 0.3 pg/ml. Calculated Sensitivity: <0.3 pg/ml The sensitivity of this assay, or the Lower Limit of Detection, is the mean signal of zero + standard deviations read in dose from the standard curve. This value is the smallest dose that is not zero with 95% confidence. Calibration: The standards in this ELISA are calibrated to the NIBSC reference standard lot 89/548. One () pg of Endogen standard = NIBSC pg = 0. NIBSC units. Specificity: This ELISA is specific for the measurement of natural and recombinant human IL-. It does not cross react with human IL-α, IL-β, IL-, IL-3, IL-4, IL-5, IL-7, IL-8, IL-0, IL-, GMCSF, IFNα, IFNγ, or mouse IL-. Expected Values: A total of 40 serum and plasma samples collected from apparently healthy individuals were tested in this ELISA. The levels of human IL- found in each sample type are reported below. Sample Type Mean Median Range Serum (n=) 443 0.7 0 3,370* EDTA (n=0) 0.4 < 0.3 0. Citrate (n=8) 0.4 < 0.3 0.3 Heparin (n=0) 0.3 < 0.3 0 0.9 * IL- levels in four serum samples were greater than 300 pg/ml. IL- levels in the remaining 8 samples were < 5 pg/ml. Precision: Reproducibility of the Pierce Endogen Human IL- ELISA was evaluated in each sample matrix. To determine intra-assay precision, 0 replicates of a sample containing high levels of recombinant human IL- and 0 replicates of a sample containing low levels of recombinant human IL- were run on a single plate. Data is shown below. To evaluate inter-assay precision, samples were tested by three different operators. Each operator performed at least three separate assays on more than one day. Twelve duplicate sample values were used to calculate inter-assay precision data for each level of IL-. Data is shown below.
Intra-Assay Precision Inter-Assay Precision Sample Level Mean SD CV Mean SD CV (%) (%) Serum,.4 99. 8.,3.7 3. 0. 5.4 3.8. 9.5 8. 8.5 EDTA,33.4 75..,450.7 30.3 9.0 57. 8.4 5.3 7.5.7 9.5 Citrate,05. 9.0 5.7,50.9 44.3 9. 5. 4.7 5.7 38.5 3. 9.7 Heparin,409.3 70. 5.0,47.5 43.7 9.8 85. 7.9.8 9..5 9.8 Cell Culture,.8 0.9.3,3.3 87.7.5 Supernatant 59.0 3.5 5. 4.7 7..4 Recovery: Human serum and plasma samples and Standard Diluent controls were spiked with natural and recombinant human IL-. Expected values were calculated by adding endogenous IL- levels, from unspiked samples, to those of spiked diluent controls. Percent recovery was found by dividing observed by expected values. Level Mean Recovery Median Recovery Recovery Range Sample n = (%) (%) (%) Serum EDTA Citrate Heparin Cell Culture Supernatant,98.9 4.4,98.9 4.4,.8 7.9,.8 7.9,750.0,34. 07. 84.9 07.9 8. 9. 84.9 98. 94.7 9. 00.4 08. 88. 0.0 88.5 9.4 84. 97. 93.8 9.9 0. 97.4.4 7.5 90.4 00.3 7.4 7.8 07.5 8.8 03.4 8.7 89.4 93.5 05.9 8..8 90.0 99.8 9.8 03. Linearity of Dilution: Human serum and plasma samples spiked with human IL- and cell culture supernatants containing natural human IL- were serially diluted in Standard Diluent and evaluated in the ImmuLux Human IL- ELISA Kit. The results are shown below. Results for heparin plasma were similar to those shown for EDTA plasma. Observed values were divided by expected values to calculate % recovery and demonstrate the Linearity of Dilution of the assay. 7
Sample Dilution Expected Observed Recovery Serum EDTA Citrate Cell Culture Supernatant Technical Support Neat : :4 :8 : Neat : :4 :8 : Neat : :4 :8 : Neat : :4 :8 :,578. 789.3 394.7 97.3 98.7,800. 900. 450. 5.0.5,70.9 855.5 47.7 3.9 0.9,588.4,94. 47. 33..8,578. 784.4 397.4. 8,800. 87.7 44. 0. 3.8,70.9 880.3 40.5 95. 00.4,588.4,99.4 594.5 83. 34.3-99.4 00.7. 9. - 9.8 98. 97.9 0.0-0.9 95.0 9.3 93.9-9.7 9.9 87.5 83.0 Call Technical Assistance at 800-874-373 or 85-98-0747 or contact your local distributor. E-mail Technical Assistance at TA@piercenet.com. Online help is available. Visit our web site at. Data Template Pierce Chemical Co., 0/000. Printed in the U.S.A. 8