This immunoassay kit allows for the in vitro quantitative determination of mouse IFN-γ concentrations in serum, plasma and other biological fluids. Expiration date six months from the date of manufacture FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
2 INTRODUCTION Interferon-gamma （IFN-γ） is a dimerized soluble cytokine that is the only member of the type II class of interferons. This interferon was originally called macrophage-activating factor. The IFN-γ monomer consists of a core of six α-helices and an extended unfolded sequence in the C-terminal region. This is shown in the structural models below. The α-helices in the core of the structure are numbered 1 to 6. Full length IFN-γ is 143 amino acids in length, the models are 126 amino acids in length. Affinity for the glycosaminoglycan heparan sulfate resides solely within the deleted sequence of 17 amino acids. In contrast to interferon-α and interferon-β which can be expressed by all cells, IFN-γ is secreted by Th1 cells, Tc cells, dendritic cells and NK cells. Also known as immune interferon, IFN-γ is the only Type II interferon. It is serologically distinct from Type I interferons and it is acid-labile, while the type I variants are acid-stable. IFN-γ has antiviral, immunoregulatory, and anti-tumour properties. It alters transcription in up to 30 genes producing a variety of physiological and cellular responses. Amongst the effects are: Increase antigen presentation of macrophages. Activate and increase lysosome activity in macrophages. Suppress Th2 cell activity. Cause normal cells to increase expression of class I MHC molecules. Promotes adhesion and binding required for leukocyte migration Promotes NK cell activity. Activation by IFN-γ is achieved by its interaction with a heterodimeric receptor consisting of IFNGR1 & IFNGR2 （interferon gamma receptors）. IFN-γ binding to the receptor activates the JAK-STAT pathway. In addition, IFN-γ activates APCs and promotes Th1 differentiation by upregulating the transcription factor T-bet. IFN-γ is the hallmark cytokine of Th1 cells （whereas Th2 cells produce IL-4 and Th17 cells produce IL-17）. NK cells and CD8+ cytotoxic T cells also produce IFN-γ. IFN-γ suppresses osteoclast formation by rapidly degrading the RANK adaptor protein TRAF6 in the RANK-RANKL signaling pathway, which otherwise stimulates the production of NFκB. 3 PRINCIPLE OF THE ASSAY The microtiter plate provided in this kit has been pre-coated with an antibody specific to IFN-γ. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for IFN-γ and Avidin conjugated to Horseradish Peroxidase （HRP） is added to each microplate well and incubated. Then a TMB （3,3'5, 5' tetramethyl-benzidine） substrate solution is added to each well. Only those wells that contain IFN-γ, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of IFN-γ in the samples is then determined by comparing the O.D. of the samples to the standard curve. DETECTION RANGE 125 pg/ml-8000 pg/ml. The standard curve concentrations used for the ELISA’s were 8000 pg/ml, 4000 pg/ml, 2000 pg/ml, 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml. SPECIFICITY This assay recognizes recombinant and natural mouse IFN-γ. No significant cross-reactivity or interference was observed. SENSITIVITY The minimum detectable dose of mouse IFN-γ is typically less than 31.2 pg/ml. The sensitivity of this assay, or Lower Limit of Detection （LLD） was defined as the lowest protein concentration that could be differentiated from zero. 4 MATERIALS PROVIDED Reagent Quantity Assay plate 1 Standard 2 Sample Diluent 1 x 20 ml Biotin-antibody Diluent 1 x 10 ml HRP-avidin Diluent 1 x 10 ml Biotin-antibody 1 x 120l HRP-avidin 1 x 120l Wash Buffer 1 x 20 ml （25×concentrate） TMB Substrate 1 x 10 ml Stop Solution 1 x 10 ml STORAGE 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit, provided it is stored as prescribed above. Refer to the package label for the expiration date. 2. Opened test plate should be stored at 2-8°C in the aluminum foil bag with desiccants to minimize exposure to damp air. The kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement. 5 REAGENT PREPARATION Bring all reagents to room temperature before use. 1. Wash Buffer If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have compley dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. 2. Standard Centrifuge the standard vial at 6000-10000rpm for 30s. Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 8000 pg/ml. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard （8000 pg/ml）. The Sample Diluent serves as the zero standard （0 pg/ml）. Prepare fresh for each assay. Use within 4 hours and discard after use. 3. Biotin-antibody Centrifuge the vial before opening. Dilute to the working concentration using Biotin-antibody Diluent（1:100）, respectively. 4. HRP-avidin Centrifuge the vial before opening. Dilute to the working concentration using HRP-avidin Diluent（1:100）, respectively. Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. OTHER SUPPLIES REQUIRED Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer. An incubator which can provide stable incubation conditions up to 37°C±0.5°C. 6 SAMPLE COLLECTION AND STORAGE Serum Use a serum separator tube （SST） and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum and assay immediay or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediay or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles. Note: Grossly hemolyzed samples are not suitable for use in this assay. ASSAY PROCEDURE Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. All the reagents should be added directly to the liquid level in the well. The pipette should avoid contacting the inner wall of the well. 1. Add 100l of Standard, Blank, or Sample per well. Cover with the adhesive strip. Incubate for 2 hours at 37°C. 2. Remove the liquid of each well, don’t wash. 3. Add 100l of Biotin-antibody working solution to each well. Incubate for 1 hour at 37°C. Biotin-antibody working solution may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform. 7 4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash: Fill each well with Wash Buffer （200l） and let it stand for 2 minutes, then remove the liquid by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel. Complete removal of liquid at each step is essential to good performance. 5. Add 100l of HRP-avidin working solution to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C. 6. Repeat the aspiration and wash three times as step 4. 7. Add 90l of TMB Substrate to each well. Incubate for 10-30 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 8. Add 50l of Stop Solution to each well when the first four wells containing the highest concentration of standards develop obvious blue color. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 9. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm. CALCULATION OF RESULTS Using the professional soft "Curve Exert 1.3" to make a standard curve is recommended, which can be downloaded from our web. Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic （4-PL） curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the 8 IFN-γconcentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. LIMITATIONS OF THE PROCEDURE The kit should not be used beyond the expiration date on the kit label. Do not mix or substitute reagents with those from other lots or sources. It is important that the Standard Diluent selected for the standard curve be consistent with the samples being assayed. If samples generate values higher than the highest standard, dilute the samples with the appropriate Standard Diluent and repeat the assay. Any variation in Standard Diluent, operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding. This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Quantikine Immunoassay, the possibility of interference cannot be excluded. TECHNICAL HINTS Centrifuge vials before opening to collect contents. When mixing or reconstituting protein solutions, always avoid foaming. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision. 9 To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Substrate Solution should remain colorless or light blue until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless or light blue to gradations of blue. Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.
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