First, matters needing attention
1. This reagent is an in vitro test reagent. It should be used as an infectious agent during the efficacy period. The reagents of different total batch numbers should not be mixed.
The reagents in the cartridge should be taken out before use. Leave at room temperature for at least 30 minutes.
After crystallization of the concentrated washing solution, incubate at 37 ° C for 15 minutes.
After crystallization of the concentrated sample dilution, the human ELISA kit should be incubated at 37 ° C for 15 minutes.
If the experiment is carried out within 24 hours, the specimen can be stored at 2-8 °C. No need to experiment in time, the specimen is stored at -20 °C to avoid repeated freezing and thawing.
The microplate is repeatedly cleaned and the residual liquid in the microwell is buckled, otherwise the accuracy will be reduced, causing an artifact of the absorbance deviation.
After the addition is completed, care should be taken to gently shake the microporous reaction strip to allow the liquid in the well to mix well.
The kit should be stored at 2~8 °C, please do not freeze it. Please see the box for the validity period.
Second, preparation before the experiment
1. Remove all reagents from the box before use and leave at room temperature for at least 30 minutes.
2. Prepare various experimental instruments and materials, such as micropipettes, tips, medical distilled water, etc.
3. Concentrated lotion and medical distilled water 1..19 times diluted to become application washing liquid
4. Concentrate the sample dilution with the medical distilled water 1.. 4 times diluted into the application sample dilution
5. Dilute the sample with the application sample dilution, dilute the sample according to a volume ratio of 1:100, add 10 μl of the sample to 1 ml of the application sample dilution, and mix well for use. )
Third, the operation steps
1. Remove the microplate and set a blank well. Add 100 μl of standard in blank microwell according to the order (the blank is regarded as standard No. 0 and replaced with medical distilled water)
2. Label the sample numbers separately and add 100 μl of the diluted sample to the blank wells (different samples with different tips).
3. The enzyme plate was incubated at 37 ° C for 30 minutes;
4. Take out the microplate and remove the liquid. After filling each well with the application of the washing liquid, immediately remove the liquid;
5. After filling each well with the application of the washing liquid, gently shake the plate for 30 seconds, then remove the applied washing liquid in the hole, and pat the enzyme plate on the absorbent paper.
6. Repeat the 5th step 5 times and pat the enzyme plate on the absorbent paper.
7. Add 100 μl of the enzyme-labeled coupling solution to the standard wells and sample wells.
8. Incubate the 96-well plate for 30 minutes at 37 °C.
9. Remove the enzyme label and remove the liquid. After filling each well with the application of the washing liquid, immediately remove the liquid.
10. After filling the washing application liquid again in each well, gently shake the microplate at room temperature for 30 seconds, then remove the applied washing liquid in the well, and pat the enzyme plate on the absorbent paper.
11. Repeat the 5th step 5 times and pat the enzyme plate on the blotting paper.
12. Add 50 μl of substrate B immediately after adding 50 μl of substrate A to each well. Mix gently by shaking. (A liquid and B liquid are loaded with different tips)
13. The enzyme-labeled plate was incubated at 37 ° C for 15 minutes in the dark.
14. Add 50 μl of stop solution to each well and mix gently by shaking.
15. Determine the OD at 450 nm on a microplate reader; after color development, measure within 15 minutes.
16. Calculate the sample content according to the prepared standard curve human ELISA kit.
Fourth, the results of judgment
1. Instrument value: read the OD value of each well on a microplate reader with a wavelength of 450 nm;
2. Detection range: 0-400pmol/L
3. Sensitivity: 1.0pmol/L
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