Elisa kit operation steps and precautions
Elisa kit procedure
1. Dilution and loading of standard products: 10 holes of standard wells are placed on the enzyme labeling plate , and 100 μl of the standard is added to the first and second holes, respectively , and then added in the first and second holes. Mix the standard dilution 50 μl and mix it; then add 100 μl from each of the first and second wells to the third and fourth wells, and then add the standard dilution in the third and fourth wells. 50 μ l solution, mix; and in the third hole and the fourth hole to discard from each 50 μ l, 50 μ l from each another are applied to the fifth and sixth holes, and then the fifth, sixth hole each with standard dilution 5OuI, mix; 50 μ l from each of the fifth and the sixth well was added to the seventh, eighth holes each, were then added in the seventh, eighth hole standard dilution 50 μ l, 50 μ l after mixing taking from the seventh, eighth holes each added to the ninth, tenth hole, then standard dilution were added 50 μ l in the ninth or tenth hole After mixing, take 50 μl from each of the ninth and tenth holes and discard. (The amount of each well was 50 μl after dilution , and the concentrations were 1200 pg/ml , 800 pg/ml , 400 pg/ml , 200 pg/ml , 100 pg/ml ).
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), and the sample holes to be tested . Add 40 μl of the sample dilution to the sample well to be tested on the plate , 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 (20 times the 48T) fold concentrated fold diluted solution was washed with distilled water and 30 alternate (48T of 20 times).
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. Â
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. Â The measurement should be carried out within 15 minutes after the addition of the stop solution .
Elisa kit notes:
1. The kit should be taken out from the refrigerated environment and should be used at room temperature for 15-30 minutes before use. If the enzyme label is unsealed after opening, the slats should be stored in a sealed bag.
2. Washing buffer may be crystallized, can be heated in a water bath solubilization dilution, washing does not affect the results.
3. The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun.
4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the first hole of the standard well ), please first dilute the sample dilution with a certain multiple ( n times) and then measure it. When calculating, multiply the total dilution by the total dilution. Multiple ( × n × 5 ).
5. The sealing film is intended for single use only to avoid cross-contamination.
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