Dog sPDL1 ELISA Kit

Specimen requirements

1. Specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to relevant literature.
Experiments should be carried out as soon as possible after extraction.
If the test cannot be carried out immediately, the specimen can be placed in -20℃ Store, but avoid repeated freezing and thawing
2. Unable to detect containing NaN3 Samples, due to NaN3 Inhibition of horseradish peroxidase ( HRP ) activity.

Operation steps

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

2. Sample addition: Blank wells (no sample and enzyme labeled reagent are added to the blank control wells, and the other steps are the same), standard wells and sample wells to be tested are set respectively.
Accurate spike of standard on enzyme-labeled coated plate 50μl , first add sample diluent to the sample well to be tested 40μl , then add the sample to be tested 10μl (The final dilution of sample was 5 Times).
Add the sample to the bottom of the well of the enzyme labeled plate, try not to touch the well wall, and gently shake and mix well.
3. Incubation: After plate sealing with plate sealing film 37℃ Incubation 30 Minutes.
4. Solution preparation: will 30 Distilled water for double concentrated washing liquid 30 Time dilution for use
5. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with washing liquid, and let stand 30 Discard it in seconds and repeat it 5 Times, pat dry.
6. Enzyme addition: enzyme labeled reagent is added to each well 50μl , except for blank holes.
7. Incubation: Same procedure 3 。
8. Washing: Same operation 5 。
9. Color development: Add color developer to each well first A50μl And then adding a color developer B50μl , gently shake and mix evenly, 37℃ Color development protected from light 10 minute .
10. Stop: Add stop solution per well 50μl , terminate the reaction (blue immediately turns yellow at this time).
11. Measurement: Zero adjustment with blank holes, 450nm The absorbance of each well was measured sequentially by wavelength ( OD Value).
The determination shall be performed after addition of the stop solution 15 Within minutes.

calculate

Taking the concentration of the standard as the abscissa, OD The value is the ordinate, draw a standard curve on the coordinate paper, and according to the sample OD The corresponding concentration is found out from the standard curve;
Multiply by the dilution factor;
Or use the concentration of the standard substance to OD The linear regression equation of the standard curve is calculated from the value, and the sample OD Substitute the value into the equation, calculate the sample concentration, and multiply it by the dilution factor,
That is, the actual concentration of the sample.

Standard curve