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Mouse thyroxine (T4) elisa kit instruction manual
Mouse thyroxine (T4) elisa kit instruction manual
purpose of usage
This kit is used to determine the content of thyroxine (T4) in serum, plasma and related fluid samples of mice.
Experimental principle
The kit uses a double antibody sandwich method to determine the level of mouse thyroxine (T4) in the specimen. The microporous plate was coated with purified mouse thyroxine (T4) antibody to prepare a solid phase antibody, and thyroxine (T4) was sequentially added to the microcapsule of the coated monoclonal antibody, followed by HRP-labeled thyroxine (T4) antibody. The antibody-antigen-enzyme-labeled antibody complex is formed by binding, and after thorough washing, the substrate TMB is added for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The color depth is positively correlated with thyroxine (T4) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of mouse thyroxine (T4) in the sample was calculated from a standard curve.
Specimen requirements
1. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the 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 °C, but repeated freezing and thawing should be avoided;
2. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity.
Steps
Dilution of standards:
Adding samples: blank holes (the blank control wells are not added with the sample and the enzyme standard reagent, the other steps are the same), the standard holes, and the sample holes to be tested. Accurately load 50 μl of the standard on the enzyme-labeled plate, add 40 μl of the sample dilution to the well to be tested, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake it gently to mix;
Incubation: the plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes;
Solution: 30 times concentrated washing solution is diluted with distilled water 30 times and used for later use;
Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing liquid, let stand for 30 seconds, then discard it, repeat 5 times, pat dry;
Add enzyme: 50 μl of enzyme labeling reagent per well, except for blank wells;
Incubation: operation is the same as 3;
Washing: operation is the same as 5;
Color development: Add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake and mix, and develop color at 37 ° C for 15 minutes.
Termination: 50 μl of stop solution was added to each well to terminate the reaction (when the blue color turned yellow);
Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution.