Chicken triiodothyronine elisa kit instructions

Chicken triiodothyronine elisa kit instructions

Chicken elisa kit experimental principle

This kit uses the double antibody sandwich method to determine the level of chicken triiodothyronine (T3) in the specimen . The microporous plate was coated with purified chicken triiodothyronine (T3) antibody to prepare a solid phase antibody, and triiodothyronine (T3) was sequentially added to the microcapsules of the coated monoclonal antibody , and then labeled with HRP. The triiodothyronine (T3) antibody binds to form an antibody - antigen - enzyme - labeled antibody complex, which is thoroughly washed and then added to the substrate TMB for color development. TMB in HRP enzyme blue, yellow and eventually converted to the action of an acid. The color depth is positively correlated with triiodothyronine (T3) in the sample . The absorbance ( OD value) was measured at 450 nm using a microplate reader , and the concentration of chicken triiodothyronine (T3) in the sample was calculated from the standard curve .

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.

Chicken elisa kit operation steps

1. Dilution of standard: This kit provides one original standard, which can be diluted in a small tube according to the following chart.

2. Loading: Set blank holes separately (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 on the standard coated 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 sample to the bottom of the wells, and try not to touch the wall of the hole and mix gently.

3. Incubation: After sealing plate sealing film and incubated at 37 ℃ board 30 minutes.

4. Dosing: 30 times concentrated washing solution diluted with distilled water 30 times and used

5. 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, and pat dry.

6. Add enzyme: Add 50 μl of enzyme labeling reagent to each well , except for blank wells .

7. Incubation: The operation is the same as 3 .

8. Washing: The operation is the same as 5 .

9. Color development: Add A50 μ l of color developer to each well , then add B50 μ l of color developer , mix gently by shaking, and color for 15 minutes at 37 °C.

10. Termination: 50 μl of stop solution was added to each well to terminate the reaction (when the blue color turned yellow) .

Determination: The blank as zero, 4 50 nm wavelength sequentially measuring absorbance (OD) of each well.   The measurement should be carried out within 15 minutes after the addition of the stop solution .

White Willow Bark Extract