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Principle
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This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific for mouse T4 has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled mouse T4 and unlabeled mouse T4 (Standards or samples) with the pre-coated antibody specific for mouse T4. After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of T4 in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of T4 in the sample.
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Protocol
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In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the log of concentration of the standard (Y-axis), although concentration is indeed the independent variable while O.D. value is the dependent variable. The O.D. values of the standard curve may vary according to the conditions of assay performance (e.g. operator, pipetting technique, washing technique or temperature effects). This curve is provided for demonstration only. The customers should establish their own standard curve for each test conducted. Typical Standard Curve for Mouse T4 ELISA.
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Reagent Preparation
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1. Bring all kit components and samples to room temperature (18-25°C) before use. 1. 2. Standard - Reconstitute the Standard with 0.5mL of Standard Diluent, kept for 10 minutes at room temperature, shake gently (not to foam). The concentration of the standard in the stock solution is 240ng/mL. Please prepare 5 tubes containing 0.6mL Standard Diluent and produce a triple dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Set up 5 points of diluted standard such as 240ng/mL, 80ng/mL, 26.67ng/mL, 8.89ng/mL, 2.96ng/mL, and the last EP tubes with Standard Diluent is the blank as 0ng/mL. Tube 1 2 3 4 5 6 ng/mL 240 80 26.67 8.89 2.96 0 3. Assay Diluent A and Assay Diluent B - Dilute 6mL of Assay Diluent A or B Concentrate(2x) with 6mL of deionized or distilled water to prepare 12 mL of Assay Diluent A or B. The prepared working dilution can't be frozen. Detection Reagent A and Detection Reagent B - Briefly spin or centrifuge the stock Detection A and 4. Detection B before use. Dilute to the working concentration with working Assay Diluent A or B, respectively (1:100). 5. Wash Solution - Dilute 20mL of Wash Solution concentrate (30x) with 580mL of deionized or distilled water to prepare 600 mL of Wash Solution (1x). 6. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again. Note: 1. Making serial dilution in the wells directly is not permitted. Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37 °C directly. 2. 3. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10L for once pipetting. 4. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once once. 5. If crystals have formed in the Wash Solution concentrate (30x), warm to room temperature and mix gently until the crystals have completely dissolved. 6. Distilled water is recommended to be used to make the preparation for reagents or samples. Contaminated water or container for reagent preparation will influence the detection result.
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Sample Collection
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Serum - Use a serum separator tube and allow Samples to clot for two hours at room temperature or overnight at 4 C before centrifugation for 20 minutes at approximately 1000xg. Assay freshly prepared serum immediately or store Samples in aliquot at -20°C or -80 C for later use. Avoid repeated freeze/thaw cycles. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge Samples for 15 minutes at 1000xg at 2 - 8 C within 30 minutes of collection. Remove plasma and assay immediately or store Samples in aliquot at -20°C or -80 C for later use. Avoid repeated freeze/thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 g. Remove particulates and assay immediately or store samples in aliquot at -20 or -80°C for later use. Avoid repeated freeze/thaw cycles.
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Sample Preparation
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The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance. 2. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments. 3. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary. 4. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts of certain chemicals. 5. Owing to the possibility of mismatching between antigen from other resource and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products. 6. Influenced by the factors including cell viability, cell number and also sampling time, samples from cell culture supernatant may not be detected by the kit. 7. Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.
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Assay Procedure
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1. Determine wells for diluted standard, blank and sample. Prepare 5 wells for standard, 1 well for blank. Add 50L each of dilutions of standard (read Reagent Preparation), blank and samples into the appropriate wells, respectively. And then add 50L of Detection Reagent A to each well immediately. Shake the plate gently. Cover with a Plate sealer. Incubate for 1 hour at 37 °C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform. 2. Aspirate the solution and wash with 350L of 1X Wash Solution to each well using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1-2 minutes. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Repeat 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper. Add 100L of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37 °C after 3. covering it with the Plate sealer. 4. Repeat the aspiration/wash process for five times as conducted in step 2. 5. Add 90L of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 25 minutes at 37 °C (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate Solution. 6. Add 50L of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 7. Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the surface of the liquid. Then, run the microplate reader and conduct measurement at 450nm immediately. Note: 1. Assay preparation: Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20 °C until the kits expiry date. Samples or reagents addition Please use the freshly prepared Standard. Please carefully add samples 2. additionPlease to wells and mix gently to avoid foaming. Do not touch the well wall as possible. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and specimens, although not required, is recommended. 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. 3. Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation time and temperature must be observed. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential to good 4. performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor precision and falsely elevated absorbance reading. 5. Controlling of reaction time: Observe the change of color after adding TMB Substrate (e.g. observation , once every 10 minutes), if the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading. 6. TMB Substrate is easily contaminated. Please protect it from light.
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Calculation of Results
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This assay employs the competitive inhibition enzyme immunoassay technique, so there is an inverse correlation between T4 concentration in the sample and the assay signal intensity. Low levels of T4 result in a high O.D value, while a high concentration of T4 results in a low signal. Average the duplicate readings for each standard, control, and samples. Create a standard curve on log-log graph paper, with T4 concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the standard points and it can be determined by regression analysis. Using some plot software, such as curve expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
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Application Notes
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1. Prepare all reagents, samples and standards, 2. Add 50L standard or sample to each well. And then add 50L prepared Detection Reagent A immediately. Incubate 1 hour at 37 C, 3. Aspirate and wash 3 times, 4. Add 100L prepared Detection Reagent B. Incubate 30 minutes at 37 C, 5. Aspirate and wash 5 times, 6. Add 90L Substrate Solution. Incubate 15-25 minutes at 37 C, 7. Add 50L Stop Solution. Read at 450 nm immediately.
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Components
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Reagents (Quantity): Pre-coated, ready to use 96-well strip plate (1), Plate sealer for 96 wells (4), Standard (freeze dried) (2), Standard Diluent (1x20ml), Detection Reagent A (green) (1x120µl), Assay Diluent A (2 x concentrate) (1x6ml), Detection Reagent B (red) (1x120µl), Assay Diluent B (2 x concentrate) (1x6ml), TMB Substrate (1x9ml), Stop Solution (1x6ml), Wash Buffer (30 x concentrate) (1x20ml)
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Material not included
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1. Microplate reader with 450 ± 10 nm filter. 2. Precision single and multi-channel pipettes and pipette tips with disposable tips. 3. Eppendorf Tubes for diluting samples. 4. Deionized or distilled water. 5. Absorbent paper for blotting the microtiter plate. 6. Container for Wash Solution
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Storage
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All the reagents should be kept according to the labels on vials. The Standard Detection Reagent A, Detection Standard, Reagent B and the 96-well strip plate should be stored at -20°C upon being received. The unused strips should be kept in a sealed bag with the desiccant provided to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit (six months from the date of manufacture). Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.
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Research Area
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Hormones
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Restrictions
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For Research Use only
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Alternatives 
