Elisa kit instructions porcine somatotropin
Experimental principle
The kit uses a double antibody sandwich method to determine the level of porcine growth hormone (GH) in the specimen . The microporous plate was coated with purified porcine growth hormone (GH) antibody to prepare a solid phase antibody, and growth hormone (GH) was sequentially added to the microcapsules of the coated monoclonal antibody , and then combined with HRP- labeled growth hormone (GH) antibody. The antibody - antigen - enzyme - labeled antibody complex was formed , and after thorough washing, the substrate TMB was added 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 growth hormone (GH) in the sample . The absorbance ( OD value) was measured at 450 nm using a microplate reader , and the concentration of porcine growth hormone (GH) in the sample was calculated from a standard curve .
Kit composition
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.
3 . Tissue treatment: After cutting the specimen, weigh the weight. Add a certain amount of PBS , pH 7.4 . It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 °C after melting . Add a certain amount of PBS ( pH 7.4 ) and homogenize the specimen by hand or homogenizer. Centrifugation 20 min (2000-3000 rev / min). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use.
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 .
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