Instructions for mouse triiodothyronine (T3)
Instruction Manual of Mouse Triiodothyronine (T3) Quantitative Detection Kit (ELISA)
ã€Kit name】 Quantitative detection kit for mouse triiodothyronine (T3) (ELISA)
[Use of the kit] Quantitatively detect the content of triiodothyronine (T3) in mouse serum, plasma and related liquid samples.
[Detection principle] This kit adopts double antibody two-step sandwich enzyme-linked immunosorbent assay (ELISA). Add the standard and the test sample to the pre-coated mouse triiodothyronine (T3) polyclonal antibody transparent enzyme label coated plate. After incubating for a sufficient time, wash to remove unbound components, and then add the enzyme Standard working solution, after incubation for a sufficient time, wash to remove unbound components. Substrates A and B were added in sequence. The substrate (TMB) was converted into a blue product under the catalysis of horseradish peroxidase (HRP), which turned yellow under the action of acid. The concentration of ortholine (T3) was positively correlated. The OD value was measured at a wavelength of 450 nm. Based on the OD value of the standard and the sample, the content of mouse triiodothyronine (T3) in the sample was calculated.
[Kit composition] 1 enzyme-coated plate 12 wells × 8 strips 7 Developer A solution 6mL 2 Standard: 8pg / mL 0.6mL 8 Developer B solution 6mL 3 20-fold concentrated washing solution 25mL 9 Stop solution 6mL 4 Standard Diluent 6mL 10 Instructions 1 copy 5 Sample diluent 6mL 11 Sealing film 2 sheets 6 Enzyme label reagent 6mL 12 Sealed bag 1 Remarks: Standard dilutions with standard dilutions are: 8, 4, 2, 1, 0.5 , 0.25pg / mL
[Reagents and equipment needed but not provided] 1. 37 ° C incubator 2. Standard microplate reader 3. Precision pipette and disposable pipette tip 4. Distilled water 5. Disposable test tube 6. Absorbent paper
[Operating steps] 1. Preparation: Remove the reagent kit from the refrigerator and re-equilibrate at room temperature for 30 minutes. 2. Mixing solution: dilute the 20-fold concentrated washing solution with distilled water to the original one. 3. Add Standard Products and samples to be tested: take a sufficient number of enzyme-coated plates and fix them on the frame, set up standard holes, 2 test sample holes and blank control holes, record the positions of each Add 50μL of the standard product to the sample; add 10μL of the sample to be tested to the sample well, and then add 40μL of the sample diluent (that is, the sample is diluted 5 times); the blank control well is not added. 4. Incubation: Incubate in a 37 ° C water bath or thermostat for 30 minutes. 5. Wash the plate: discard the liquid, pat dry on the absorbent paper, fill each well with washing liquid, let stand for 1min, shake off the washing liquid, pat dry on the absorbent paper, repeat washing the plate 4 times (you can also use the washing machine to press Instructions for washing the board). 6. Add enzyme-labeled working solution: add 50μL of enzyme-labeled working solution to each well, without adding blank control wells. 7. Incubation: Repeat 4 operations. 8. Wash the board: repeat the operation of 4. 9. Color development: add 50μL of developer A solution to each well, then add 50μL of developer B solution, and develop color at 37 ° C in the dark for 15min. 10. Termination: Remove the enzyme labeling plate and add 50μL of stop solution to each well to stop the reaction (the color changes from blue to yellow). 11. Determination: Zero the blank holes, and within 15 minutes after termination, measure the absorbance (OD value) of each well with a wavelength of 450 nm. 12. Calculation: According to the concentration of the standard product and the corresponding OD value, calculate the linear regression equation of the standard curve, and then calculate the corresponding sample concentration on the regression equation according to the OD value of the sample. You can also use various application software to Calculation. The final concentration is the actual measured concentration times the dilution factor.
[Sample requirements] 1. The sample must not contain sodium azide (NaN3), because sodium azide (NaN3) is an inhibitor of horseradish peroxidase (HRP). 2. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided. 3. The sample should be fully centrifuged, without hemolysis and particles.
[Notes] 1. The experiment is carried out in strict accordance with the instructions, and the result of the experiment must be determined by the reading of the microplate reader. 2. If the enzyme-labeled coated board is not used up after opening, it should be put into a sealed bag and added with desiccant immediately. 3. It is recommended that all standards, samples and blank controls be tested in duplicate, and the average value is taken to reduce the experimental error. 4. Keep in mind that the sample has been diluted 5 times, and the calculation result is multiplied by 5 to obtain the actual concentration of the sample. 5. The quantitative range of this kit is 0.25-8pg / mL, beyond this range, it is calculated from the extension of the standard curve, not as an accurate quantitative result, please dilute it with a special diluent to determine the accurate result (within the range of 0.25-8pg / mL) ), Multiplied by the total dilution factor is the final concentration of the sample. 6. If the color is too light, the substrate incubation time can be extended properly. 7. In order to avoid cross-contamination, the tips, samples and blank controls should be replaced with a new one for each addition; the common components such as enzyme working solution, sample dilution and substrate should be cantilevered and do not touch the microwells. ; Do not reuse the sealing film. 8. The kits are used within the warranty period, and different batches of reagents should not be mixed. 9. Substrate B is sensitive to light and avoid prolonged exposure to light.
[Summary of operating procedures] 3 Prepare reagents, add samples and standards to the prepared samples and standards, wash the plate 4 times at 37 ° C for 30 minutes, add enzyme reagent, wash the plate 4 times at 37 ° C for 30 minutes, add color Solution A, B, color development at 37 ° C for 15 minutes, add stop solution and read OD value within 15 minutes
ã€Detection range】 0.25-8pg / mL
ã€Specifications】 96 servings / box
[Storage] Store at 2-8 ℃, protected from light and moisture.
[Validity period] 6 months Shanghai Yuping Biological Technology Co., Ltd. mainly deals with ELISA kits of various brands and grades, with quality assurance and perfect after-sales service. And provide free generation testing. Serving universities and immunology research units. Technicians serve you better.
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