Porcine tumor necrosis factor alpha (TNF-α) elisa technique

Porcine tumor necrosis factor alpha (TNF-α) elisa technology method Guangrui Bio- domestic high and excellent Elisa kit supplier steps: 1. Dilution and loading of standard products: standard wells on the enzyme label coating board 10 wells, add 100 μl of standard in the first and second wells, then add 50 μl of the standard dilution in the first and second wells, mix; then take 100 μl from each of the first and second wells. Add to the third hole and the fourth hole, and then add 50 μl of the standard dilution solution to the third and fourth holes, and mix; then, take 50 μl in the third hole and the fourth hole, discard each, and then take 50 μl each. In the fifth and sixth holes, add 50 ul of the standard dilution solution in the fifth and sixth holes, and mix; after mixing, take 50 μl from each of the fifth and sixth holes and add to the seventh and the sixth respectively. In the eight wells, add 50μl of the standard dilution solution to the seventh and eighth wells. After mixing, take 50μl from the seventh and eighth holes, add to the ninth and tenth holes, and then in the ninth. Add 50 μl of the standard dilution to each of the ten wells, and mix and discard 50 μl from each of the ninth and tenth holes. (The amount of each well was 50 μl after dilution, and the concentrations were 240 ng/L, 160 ng/L, 80 ng/L, 40 ng/L, 20 ng/L, respectively). 2. Adding samples: 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), and the sample holes to be tested. Add 40 μl of the sample dilution to the sample well to be tested on the plate, 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 gently to mix. 3. Incubation: After sealing with a sealing film, incubate at 37 ° C for 30 minutes. 4. Dosing: Dilute 30 (20 times of 48T) concentrated washing solution with distilled water 30 (20 times of 48T) and set aside. 5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, 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: operation is the same as 3. 8. Washing: operation is the same as 5. 9. Color development: add 50 μl of color developer A, add 50 μl of color developer B, gently shake and mix, and avoid light at 37 °C. 15 min.10. Termination: 50 μl of stop solution was added to each well to stop the reaction (the blue color turned yellow). 11. 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.

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