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Prinzip
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This assay employs the competitive inhibition enzyme immunoassay technique. A polyclonal antibody specific for human T4 has been pre-coated onto a microplate. A competitive inhibition reaction is launched between with biotin labeled human T4 and unlabeled human T4 (Standards or samples) with the pre-coated antibody specific for human T4. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well and a change in color will be exhibited. The more the amount of human T4 in samples, the less the biotin labeled human T4 bound by pre-coated antibody. The substrate solution are added to the wells, respectively. And the color develops in opposite to the amount of human T4 bound in the initial step. The color development is stopped and the intensity of the color is measured.
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Aufbereitung der Reagenzien
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1. Bring all kit components and samples to room temperature (18-25 °C ) before use. 2. Standard - The concentration of the standard is as following: 320 ng/mL, 160ng/mL, 80 ng/mL, 40 ng/mL, 20 ng/mL, 0 ng/mL. 3. Wash Solution - Dilute 15mL of Wash Solution Concentrate(20 ) with 285ml of deionized or distilled water to prepare 300 mL of Wash Solution(1 ). If crystals have formed in the Wash Solution concentrate(20 ), warm to room temperature and mix gently until the crystals have completely dissolved. 4. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.
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Probennahme
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Sample 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 1000 g. Assay freshly prepared serum immediately or store Samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles. Note: 1. Samples to be used within 5 days may be stored at 2-8 °C , otherwise Samples must stored at -20 °C ( 1 month) or -80 °C ( 2 months) to avoid loss of bioactivity and contamination. 2. When performing the assay slowly bring Samples to room temperature. 3. Sample hemolysis will influence the result, so hemolytic specimen can not be detected.
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Testdurchführung
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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. 1. Determine wells for blank, diluted standard, and sample. Prepare 5 wells for standard, 1 well for blank and other wells for sample. Add 50 each of standard solution(see Reagent Preparation 2 ) and samples into the appropriate wells, respectively. And then add 50 of Detection Reagent A to each tube immediately. Shake the plate gently. Cover with a Plate sealer. Incubate for 1 hour at 37 °C°C. 2. Aspirate the solution and wash with about 300-350 of 1X Wash Solution to each well using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher, and let it sit for 10 seconds. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Repeat 3-5 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper. 3. Add 50 of Detection Reagent B to each well. Shake the plate gently. Cover with a new Plate sealer. Incubate for 30 minutes at 37 °C . 4. Repeat the aspiration/wash process for 3-5 times as conducted in step 2. 5. Add 90 of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 minutes at 37 °C . Protect from light. 6. Add 50 of Stop Solution to each well to stop the reaction. 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 4 until the kits expiry date. 2. Samples or reagents addition Please carefully add samples to wells and mix gently addition 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. 4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential to good 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 colour after adding TMB , Substrate (e.g. observation once every 5 minutes), if the colour 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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Ergebnisberechnung
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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 luminescence intensity, 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 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 x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph (5 points). The data may be linearized by plotting the log of the T4 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
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Bestandteile
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Reagents (Quantity): Pre-coated, ready to use 96-well strip plate (1), Standard-1,2,3,4,5,6 6 Detection Reagent A (green) 1× 6ml Detection Reagent B (red) 1× 6ml TMB Substrate 1× 9ml Stop Solution 1× 6ml Wash Buffer(20 x concentrate) 1 ×15ml Plate sealer for 96 wells (4)
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Benötigtes Material
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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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Lagerung
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The kits should be stored at 2-8 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 anufacture). Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.
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Forschungsgebiet
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Hormone
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Beschränkungen
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Nur für Forschungszwecke einsetzbar
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Alternativen 
