Experimental reagent

1. Cholecystokinin (CCK) quantitative detection kit (ELISA)

2. Distilled water

Laboratory equipment

1. 37 ° C incubator

2. Standard specification microplate reader

3. Precision pipettes and disposable tips

4. Disposable test tube

5. Absorbent paper

Experimental procedure

1. Preparation: Remove the kit from the refrigerator and equilibrate for 30 minutes at room temperature.

2. Dosing: Dilute 20 times concentrated washing solution with distilled water to the original washing solution.

3. Add standard and sample to be tested: Take a sufficient number of enzyme label coated plates, fix them on the frame, set standard wells, sample holes to be tested and blank control holes, record the position of each hole, in the standard hole Add 50 μL of the standard sample; add 10 μL of the sample to be tested first, and then add 40 μL of the sample dilution (ie, the sample is diluted 5 times); the blank control well is not added.

4. Incubation: Incubate for 30 min at 37 °C in a water bath or incubator.

5. Wash the board: discard the liquid, pat dry on the absorbent paper, fill each well with the washing liquid, let stand for 1 min, remove the washing liquid, pat dry on the absorbent paper, and repeat the washing 4 times (you can also use the washing machine to press Instructions for washing the board).

6. Add the enzyme standard working solution: add 50 μL of the enzyme standard working solution to each well, and the blank control well is not added.

7. Incubation: Incubate for 30 min at 37 °C in a water bath or incubator.

8. Wash the board: discard the liquid, pat dry on the absorbent paper, fill each well with the washing liquid, let stand for 1 min, remove the washing liquid, pat dry on the absorbent paper, and repeat the washing 4 times (you can also use the washing machine to press Instructions for washing the board).

9. Color development: add 50 μL of developer A solution to each well, then add 50 μL of developer B solution, mix for 30 seconds with a plate mixer (or gently shake for 30 seconds), and avoid coloration for 15 minutes at 37 °C. .

10. Termination: Remove the plate, add 50 μL of stop solution to each well, and stop the reaction (the color turns from blue to yellow).

11. Measurement: Zeroing was performed with a blank hole, and the absorbance (OD value) of each well was measured with a wavelength of 450 nm within 15 minutes after termination.

12. Calculation: Calculate the linear regression equation of the standard curve according to the concentration of the standard and the corresponding OD value, and then calculate the corresponding sample concentration on the regression equation according to the OD value of the sample. You can also use various application software to Calculation. The final concentration is the actual measured concentration multiplied by the dilution factor.

Precautions

1. The sample should not contain sodium azide (NaN3) because sodium azide (NaN3) is an inhibitor of horseradish peroxidase (HRP).

2. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If it is not possible to test immediately, the specimen can be stored at -20 ° C, but repeated freezing and thawing should be avoided.

3. The sample should be fully centrifuged without hemolysis or granules.

4. The experiment is carried out in strict accordance with the operation of the manual. The results of the experiment must be determined by the reading of the microplate reader.

5. If the enzyme label is not used up after being opened, it should be immediately placed in a sealed bag and desiccant.

6. It is recommended that all standards, samples, and blanks be double-tested and averaged to reduce experimental error.

7. Keep in mind that the sample has been diluted 5 times and the calculated result is multiplied by 5 to determine the actual concentration of the sample.

8. The kit has a quantitative range of 20-640ng/L. If it exceeds this range, it is calculated by the extension of the standard curve. It is not used as an accurate quantitative result. Please dilute with a special dilution solution to determine the accurate result (20-640ng/L). ), multiplied by the total dilution factor is the final concentration of the sample.

9. If the color is too light, extend the substrate incubation time appropriately.

10. In order to avoid cross-contamination, the standard, sample and blank control should be replaced once for each additional one; the common components such as enzyme standard working solution, sample diluent and substrate should be cantilevered and should not touch micropores. The sealing film must not be reused.

11. The kits should be used during the shelf life. Different batches of reagents should not be mixed.

12. Substrate B is sensitive to light and avoids prolonged exposure to light.

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