Human APPβ 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: Set up 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 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: zeroing 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.

1. The kit should be taken out of the refrigerated environment and balanced at room temperature for 1 hour before it can be used. If the enzyme-labeled coated plate is not used up after opening, the strips should be stored in a sealed bag.

2. Crystals may precipitate in the concentrated washing liquid. When diluting, it can be heated in a water bath to help dissolve, and the results will not be affected during washing.

3. A sampler should be used in each step of sampling, and its accuracy should be checked frequently to avoid test errors. It is best to control the sample addition time within 5 minutes. If the number of specimens is large, it is recommended to use a row gun to add samples.

4. Please make a standard curve at the same time of each measurement, preferably a double hole. If the content of the substance to be tested in the sample is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please dilute it with the sample diluent for a certain multiple (n times) before measuring, and please finally multiply it by the total dilution multiple (× n × 5) when calculating.

5. The sealing film is only for one-time use to avoid cross-contamination.

6. Please keep the substrate away from light.

7. Strictly follow the instructions, and the test results must be determined based on the reading of the microplate reader.

8. All samples, washing solutions and various wastes should be treated as infectious agents.

9. Components of different batch numbers of this reagent shall not be mixed.