Protocol VeriKine TM Human Interferon Omega ELISA Kit Catalog No: 41395 Lot No: Expiration: Assay Range: 4.7-300 pg/ml Store all components at 2 8 C Please review the protocol in its entirety prior to use to ensure proper kit performance. Sold under license from Pestka Biomedical Laboratories, Inc. d/b/a PBL Assay Science. For research use only. Not for diagnostic or clinical use in, or administration to, humans. Not for resale in original or any modified form, including inclusion in a kit, for any purpose. Not for use in the preparation of any commercial product. Copyright 2013 Pestka Biomedical Laboratories, Inc. All rights reserved. Materials Provided: Additional Materials Required (NOT PROVIDED): Pre-coated microtiter plate Plate sealers Biotin-Conjugate Streptavidin-HRP IFN Omega Standard Concentrate, 600 pg/ml Wash Buffer Concentrate Assay Buffer Concentrate Sample Diluent Substrate Solution Stop Solution Microtiter plate reader capable of reading an OD at a wavelength of 450 nm Variable volume microtiter pipettes Adjustable multichannel pipette (50-300 µl) Reagent reservoirs Wash bottle or plate washing system Distilled or deionized water Serological pipettes (1, 5, 10 or 25 ml) Disposable pipette tips (polypropylene) Plate shaker Kit Components Lot No. Quantity Plate 1 Plate Sealers N/A 4 Biotin-Conjugate* 200 µl; anti-ifn-ω monoclonal (murine) antibody Streptavidin-HRP* 150 µl IFN-ω Standard Concentrate* dissolve in 450 µl 600 pg/ml upon dilution (2 vials) Wash Buffer Concentrate 50 ml Assay Buffer Concentrate* 5 ml Sample Diluent 12 ml Substrate Solution 15 ml Stop Solution 15 ml *Reagents contain preservatives Specifications: This kit quantitates human interferon omega in cell culture supernatants, human serum, plasma or other body fluids using a sandwich immunoassay. 1,2 The kit is an ELISA that uses a biotinylated antibody and streptavidin conjugated horseradish peroxidase (HRP). Tetramethyl-benzidine (TMB) is the substrate. All reagents are supplied. One pre-coated microtiter plate (96 wells) is included. Typical standard curves for each lot are included with the procedure. Speed: Incubation time, 3 hr 15 min Page 1 of 7
Specificity: Human IFN-ω. No cross reactivity detected with human IFN-γ, human IFN-β or human IFN-α. Storage Conditions/Comments: For retention of full activity, all reagents should be kept at 2-8 C in the dark. Deionized or distilled water should be used for preparation of the Assay Buffer and Wash Buffer. All dilutions should be made with polypropylene tubes and pipette tips. Pipette tips should be changed between each dilution tube. All measurements for standards and samples should be performed in duplicate. At least two control wells (wells with Dilution Buffer only) should be used for each assay; these control values should be subtracted from all readings prior to any calculations or plots of the data. Specimen Collection: Cell culture supernatants, human serum, EDTA, heparin, and citrate plasma or other biological samples will be suitable for use in the assay. Remove serum from the clot or red blood 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 limedic specimens. Clinical samples should be kept at 2-8 C and separated rapidly before storing at -20 C to avoid loss of bioactive IFN-ω. If samples are to be run within 24 hours, they may be stored at 2-8 C. Avoid repeated freeze-thaw cycles. Assay Procedure Quick Reference Total Time: 3 hr 15 min To Standard Curve Designated Wells: 1) Add 100 µl Diluted Standard or Blank 2) Add 50 µl Biotin-Conjugate Incubate 2 hrs (shake at 400 rpm) Aspirate and wash 3x (Soak in wash solution for 10-15 seconds before aspiration) To Sample Designated Wells: 1) Add 75 µl Sample Diluent 2) Add 25 µl Prepared Sample 3) Add 50 µl Biotin-Conjugate Add 100 µl Diluted HRP Solution Incubate 1 hr (shake at 400 rpm) Aspirate and wash 3x (Soak in wash solution for 10-15 seconds before aspiration) Add 100 µl TMB Substrate Incubate 10 min in the dark Do not seal or wash. Add 100 µl Stop Solution Read plate within 5 min (450 nm) Note: All incubations are at room temperature, 22-25 C. Page 2 of 7
Preparation of Reagents Before starting the assay, the plate(s), Wash Buffer Concentrate, applicable dilution matrices, Stop Solution and samples should be equilibrated to room temperature (RT), 22-25 C. The TMB Substrate Solution should be equilibrated to RT (22-25 C) during step 3 of the Assay Procedure. Supplied Human IFN Omega Standard, Biotin-Conjugate, Streptavidin-HRP, Assay Buffer Concentrate, Sample Diluent, Substrate Solution, and dyes should be kept on ice (4 C). A. Wash Buffer: The Wash Buffer Concentrate may contain crystals. Place the bottle in a warm water bath and gently mix until completely dissolved. Prepare a 1:20 working wash buffer by adding Wash Buffer Concentrate to distilled or deionized water, according to the table below, and mix thoroughly. The ph of the final solution should adjust to 7.4. Transfer to a clean wash bottle and store at RT (2 2-25 C) when not in use. Please note that Wash Buffer (1X) is stable for 30 days. Number of Strips Wash Buffer Concentrate (ml) Distilled Water (ml) 1-6 25 475 7-12 50 950 B. Assay Buffer: Mix the contents of the bottle well. Prepare a 1:20 working assay buffer by adding Assay Buffer Concentrate to distilled or deionized water, according to the table below, and mix thoroughly. Store at 2-8 C. Please note that the Assay Buffer is stable for 30 days. Number of Strips Assay Buffer Concentrate (ml) Distilled Water (ml) 1-6 2.5 47.5 7-12 5.0 95.0 C. Human Interferon Omega Standard Solution: Add 450 µl Assay Buffer (reagent B) to one vial of concentrated IFN-ω Standard. The volume is stated on the label of the standard vial. Shake gently to mix (concentration of standard=600 pg/ml). Store diluted Standard promptly at -20 C after use. Standard Curve Preparation: Construct a standard curve 4.7-300 pg/ml in the sample matrix. a) Label polypropylene tubes as S7-S1, as indicated in Figure 1. b) Fill tubes with Sample Diluent as indicated. c) Using polypropylene tips add the diluted Human IFN Omega Standard to S7 and mix gently. Change tips between each dilution. d) Remove indicated amount from S7 and add to S6. Repeat to complete series to S1. e) Refrigerate until use in step 1 of the Assay Procedure. Figure 1: 7-Point Standard Curve Prepared in Sample Diluent Sample Matrix Label S7 S6 S5 S4 S3 S2 S1 Blank Sample Diluent Vol. (µl) 250 250 250 250 250 250 250 250 IFN-ω Conc. (pg/ml) 300 150 75 37.5 18.8 9.4 4.7 0 D. Biotin-Conjugate: Prepare a 1:100 dilution of Biotin-Conjugate with Assay Buffer (reagent B) according to the table below. Number of Strips Biotin-Conjugate (ml) Assay Buffer (ml) 1-6 0.03 2.97 7-12 0.06 5.94 Please note that Biotin Conjugate should be used within 30 minutes after dilution. Page 3 of 7
E. Streptavidin-HRP: Prepare within 30 minutes prior to use and keep on ice (4 C) until step 3 of the Assay Procedure. 1) Prepare a 1:300 dilution of Streptavidin-HRP Concentrate with Assay Buffer (reagent B) according to the table below. Number of Strips Streptavidin-HRP (ml) Assay Buffer (ml) 1-6 0.020 6 7-12 0.040 12 2) Alternatively, the Streptavidin-HRP Concentrate may be pre-diluted by adding 300 μl of Assay Buffer (reagent B) to the vial containing the concentrate. Mix the contents of the tube well. Make a further 1:100 dilution of this solution according to the table below. Number of Strips Streptavidin-HRP (ml) Assay Buffer (ml) 1-6 0.060 6 7-12 0.120 12 F. TMB Substrate Solution: Using clean pipettes and containers known to be metal free, dispense 100 μl of Substrate Solution to each well. The TMB Substrate Solution may develop a yellow tinge over time. This does not seem to affect product performance. A blue color present in the TMB Substrate Solution, however, indicates that it has been contaminated and must be discarded. The TMB Substrate Solution must be used within a few minutes after mixing. Warm to RT (22-25 C) before use. Avoid direct exposure of TMB reagents to intense light and oxidizing agents during storage or incubation. Prepare TMB Substrate according to the table below. Number of Strips Substrate Solution (ml) 1-6 6.0 7-12 12.0 Assay Procedure All incubations should be performed at RT (22-25 C), keeping the plate away from drafts and other temperature fluctuations. Use plate sealers to cover the plate as directed. During all wash steps, remove contents of plate by inverting and shaking over a sink and blotting the plate on lint-free absorbent paper; tap the plate. Wash each well with a minimum of 300 μl of diluted Wash Buffer at each wash step. See Preparation of Reagents for details on dilution of concentrated solutions. Figure 2: Example of a Typical Plate Setup B = Blank S1 S7 = Standard Curve Sa = Sample 1. Standards and Test Samples: Determine the number of microtiter plate strips required to test the desired number of samples plus the appropriate number of wells needed to run blanks and standards. We recommend running the IFN-ω Standard, blanks, samples and optional control sample in duplicate (see Figure 2 for example plate setup). Remove extra microtiter plate strips from the frame, seal in the foil bag provided and store at 2-8 C. Unused strips can be used in later assays Page 4 of 7
Wash the microtiter plate twice with approximately 300 μl Wash Buffer per well with thorough aspiration of microplate contents between washes. Allow the wash buffer to sit in the wells for about 10-15 seconds before aspiration. Take care not to scratch the surface of the microtiter plate. After the last wash, empty wells and tap the plate on lint-free absorbent paper to remove excess Wash Buffer. Use the microtiter plate strips immediately after washing or place upside down on a wet absorbent paper for no longer than 15 minutes. Do not allow wells to dry. 1a. For Standard Curve Wells: - Add 100 μl of diluted Standard or Blank (refer to Preparation of Reagents) - Add 50 μl Biotin-Conjugate to each well 1b. For Sample Wells: - Add 75 μl of Sample Diluent - Add 25 μl of Test Sample (or Sample Diluent for blank wells) - Add 50 μl Biotin-Conjugate to each well Cover with a plate sealer and shake plate at 400 rpm at RT (22-25 C) for 2 hours. After 2 hours, empty the contents of the plate and wash the wells three times with at least 300 μl of working Wash Buffer. Allow the wash buffer to sit in the wells for about 10-15 seconds before aspiration. 2. Streptavidin-HRP: Add 100 μl of diluted Streptavidin-HRP (refer to Preparation of Reagents) to each well. Cover with a plate sealer and shake plate at 400 rpm at RT (22-25 C) for 1 hour. During this incubation period, warm the TMB Substrate Solution to RT (22-25 C). After 1 hour, empty the contents of the plate and wash the wells three times with at least 300 μl of working Wash Buffer. Allow the wash buffer to sit in the wells for about 10-15 seconds before aspiration. 3. TMB Substrate Solution: Add 100 μl of the TMB Substrate Solution to each well. Incubate in the dark, at RT (22-25 C), for about 10 minutes. Do not use a plate sealer. Avoid direct exposure to intense light. The point at which the substrate reaction is stopped is often determined by the ELISA reader being used. Many ELISA readers record absorbance only up to 2.0 OD. Therefore the person running the assay must watch the color development within individual microtiter plates and have the substrate reaction stopped before positive wells are no longer properly recordable. 4. Stop Solution: After about 10 minute incubation of TMB, DO NOT EMPTY THE WELLS AND DO NOT WASH. Add 100 μl of Stop Solution to each well to stop the enzyme reaction. It is important that the Stop Solution is spread quickly and uniformly throughout the microtiter plate to completely inactivate the enzyme. Results must be read immediately after the Stop Solution is added or within one hour if the microtiter plate is stored at 2-8 C in the dark. 5. Read: Using a microplate reader, determine the absorbance at 450 nm within 5 minutes after the addition of the Stop Solution. (Optionally 620 nm can be used as the reference wavelength; 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 IFN-ω standards. Note: In case of incubation without shaking, the obtained OD values may be lower, but the results are still valid. Calculation of Results Calculate the average absorbance values for each set of duplicate standards and samples. Duplicates should be within 20 percent of the mean. Create a standard curve by plotting the mean absorbance for each standard concentration on the ordinate against the IFN-ω concentration on the abscissa. Draw a best-fit curve through the points of the graph. To determine the concentration of circulating IFN-ω 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 IFN-ω concentration. Page 5 of 7
For samples that have been diluted according to the instructions given in this manual (1:4), the concentration read from the standard curve must be multiplied by the dilution factor (x 4). Note: Calculation of samples with an OD exceeding 1.0 may result in incorrect, low IFN-ω levels. Such samples require further dilution with Sample Diluent in order to precisely quantitate the actual IFN- level. It is suggested that each testing facility establishes a control sample of known IFN-ω 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. Results of a typical standard curve are provided for demonstration only and should not be used to obtain test results. A standard curve must be run for each set of samples assayed. Figure 3: Typical Standard Curve Absorbance (450 nm) Human IFN-ω (pg/ml) Page 6 of 7
References 1. Adolf, G.R. (1987). Antigenic Structure of Human Interferon (Interferon II): Comparison with Other Human Interferons. J. Gen. Virol. 68: 1669-1676. 2. Adolf, G.R. et al (1991). Human interferon : Isolation of the gene, expression in Chinese hamster ovary cells and characterization of the recombinant protein. Biochem. et Biophys. Acta, 1089: 167-174. 3. Adolf, G.R. (1990). Monoclonal Antibodies and enzyme immunoassays specific for human interferon (IFN) 1: evidence that IFN-1 is a component of human leukocyte IFN. Virology 175: 410-417. 4. Adolf, G.R. et al (1990). Purification and characterization of Natural human Interferon l. J. Biolog. Chem. Vol. 265, 16: 9290-9295. 5. Adolf, G.R. (1995). Human interferon omega - a review. Multiple Sclerosis 1: 44-47. 6. Hauptmann R., Swetly P. (1985). A novel class of Human type I interferon. Nucl. Acids Res. 13: 4739-4749. Plate Layout Use this plate layout as a record of standards and samples assayed. Authorization Released by: Date: Sold under license from Pestka Biomedical Laboratories, Inc. d/b/a PBL Assay Science. For research use only. Not for diagnostic or clinical use in, or administration to, humans. Not for resale in original or any modified form, including inclusion in a kit, for any purpose. Not for use in the preparation of any commercial product. Page 7 of 7