Experimental reagent
1. Rat cholecystokinin (CCK) quantitative detection kit (ELISA)
2. Distilled water
experiment apparatus
1. 37 ℃ thermostat
2. Standard specification microplate reader
3. Precision pipettes and disposable tips
4. Disposable test tubes
5. Absorbent paper
Experimental procedure
1. Preparation: Remove the kit from the refrigerator and re-equilibrate at room temperature for 30 minutes.
2. Mixing solution: dilute the 20-fold concentrated washing solution with distilled water to the original one.
3. Add standard and sample to be tested: take a sufficient number of enzyme-coated plates and fix them on the frame. Set the standard well, sample well to be tested and blank control well, record the position of each well in the standard well Add 50μL of standard product; first add 10μL of sample to be tested, then add 40μL of sample diluent (that is, the sample is diluted 5 times); blank control well is not added.
4. Incubation: Incubate in a 37 ° C water bath or thermostat for 30 minutes.
5. Wash the plate: discard the liquid, pat dry on the absorbent paper, fill each well with the washing liquid, let stand for 1min, shake off the washing liquid, pat dry on the absorbent paper, repeat the washing plate 4 times (you can also use the washing machine to press Instructions for washing the board).
6. Add enzyme-labeled working solution: add 50μL of enzyme-labeled working solution to each well, without adding blank control wells.
7. Incubation: Incubate in a 37 ° C water bath or thermostat for 30 minutes.
8. Wash the plate: discard the liquid, pat dry on the absorbent paper, fill each well with the washing liquid, let stand for 1min, shake off the washing liquid, pat dry on the absorbent paper, repeat the washing 4 times (you can also use the washing machine to press Instructions for washing the board).
9. Color development: add 50μL of developer A solution to each well, and then add 50μL of developer B solution. Mix with a plate mixer for 30s (or shake gently by hand for 30s), and avoid color development at 37 ℃ for 15min .
10. Termination: Remove the microplate, add 50μL of stop solution to each well, and stop the reaction (the color changes from blue to yellow).
11. Determination: Zero the blank holes, and measure the absorbance (OD value) of each well 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 product and the corresponding OD value, and then calculate the corresponding sample concentration on the regression equation according to the OD value of the sample, or use various application software to Calculation. The final concentration is the actual measured concentration times the dilution factor.
Precautions
1. The sample cannot contain sodium azide (NaN3), because sodium azide (NaN3) is an inhibitor of horseradish peroxidase (HRP).
2. Extract the specimens as soon as possible after collection. The extraction is carried out according to relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided.
3. The sample should be centrifuged sufficiently, without hemolysis and particles.
4. The experiment is carried out in strict accordance with the instructions, and the result of the experiment must be determined by the reading of the microplate reader.
5. If the enzyme-coated plate is not used up after opening, it should be put into a sealed bag and desiccated immediately.
6. It is recommended that all standards, samples and blank controls be tested in duplicate, and the average value is taken to reduce the experimental error.
7. Keep in mind that the sample has been diluted 5 times. The calculation result is multiplied by 5 to obtain the actual concentration of the sample.
8. The quantitative range of this kit is 20-640ng / L, beyond this range, it is calculated from the extension of the standard curve. It is not used as an accurate quantitative result. Please dilute it with a special diluent to determine the accurate result (within the range of 20-640ng / L) ), Multiplied by the total dilution factor is the final concentration of the sample.
9. If the color is too light, the substrate incubation time can be extended appropriately.
10. In order to avoid cross-contamination, the tips, samples and blank controls should be replaced with a new one each time; the common components such as enzyme working solution, sample diluent and substrate should be cantilevered and do not touch the microwells. ; Do not reuse the sealing film.
11. The kit is used within the warranty period, and reagents of different batches should not be mixed.
12. Substrate B is sensitive to light and avoid prolonged exposure to light.
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