| Usage | Sample requirements:
1. The specimens were extracted as soon as possible after collection, the extraction was performed according to the relevant literature, and the experiment should be performed as soon as possible after extraction. If the test cannot be carried out immediately, the specimen can be stored at -20℃, but repeated freezing and thawing
2 should be avoided. Samples containing NaN3 could not be detected because NaN3 inhibited the activity of horseradish peroxidase (HRP).
Steps:
Dilution of standard: This kit provides one original standard, and the user can dilute it in a small test tube according to the following chart.
| 800ng/L | Standard 5 | 150uL original standard added 150uL standard dilution | | 400ng/L | Standard 4 | 150uL standard 5 add 150uL standard dilution | | 200ng/L | Standard 3 | 150uL standard 4 add 150uL standard dilution | | 100ng/L | Standard 2 | 150uL standard 3 add 150uL standard dilution | | 50ng/L | Standard 1 | 150uL of standard 2 Add 150uL of standard diluent |
2. Sample addition: set up blank Wells (blank control Wells do not add samples and enzyme-labeled reagents, the rest of the steps are the same), standard Wells, and sample Wells to be tested. On the microplate coated plate, 50uL of standard sample was accurately added, 40uL of sample diluent was added to the sample well to be tested, and then 10uL of the sample to be tested (the final dilution of the sample was 5 times). Add the sample to the bottom of the microplate hole, try not to touch the hole wall, gently shake and mix.
3. Incubation: the plate was sealed with plate sealing membrane and then incubated at 37 ° C for 30 minutes.
4. Preparation: dilute 30 times concentrated washing solution with distilled water 30 times and prepare
5. Wash: carefully remove the sealing plate membrane, discard the liquid, dry, fill each hole with washing solution, let it stand for 30 seconds, then discard, repeat 5 times, pat dry.
6. Add enzyme: add 50uL of enzyme-labeled reagent to each well, except blank well.
7. Incubation: same as 3.
8. Washing: same as 5.
9. Color development: add 50uL of color development agent A to each well first, then add 50uL of color development agent B, gently shake and mix, 37℃ dark color development for 10 minutes.
10. Termination: add 50uL termination solution to each well to terminate the reaction (at this time, the blue immediately turns yellow).
11. Determination: The blank hole was set to zero, and the absorbance (OD) value of each hole was measured sequentially at 450nm wavelength. The determination should be carried out within 15 minutes after the addition of termination solution.
Calculation:
With the concentration of the standard as the abscisordinate, OD value as the ordinate, draw a standard curve on the coordinate paper, according to the OD value of the sample to find out the corresponding concentration from the standard curve; Then multiply by the dilution factor; Or use the concentration of the standard and OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply by the dilution factor, which is the actual concentration of the sample. |
| General Notes | 1. The kit should be balanced at room temperature for 15-30 minutes before use when taken out from the cold storage environment. If the enzyme coated plate is not used up after opening, the Flat noodles should be stored in a sealed bag
2. Concentrated washing solution may have crystallization precipitation. When diluted, it can be heated in a water bath to aid in dissolution, and washing does not affect the results
3. A sampler should be used for each step of sampling, and its accuracy should be frequently checked to avoid experimental errors. It is best to control the sample addition time within 5 minutes. If there are a large number of specimens, it is recommended to use a row gun for sample addition
4. Please make a standard curve at the same time as each measurement, preferably with a re 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 the sample diluent by a certain multiple (n times) before measuring. When calculating, please multiply the total dilution multiple (× n× 5)<5. The sealing film is limited to one-time use to avoid cross contamination7. Strictly follow the instructions, and the test results must be determined based on the reading of the enzyme-linked immunosorbent assay instrument
8. All samples, washing solutions, and various waste should be treated as infectious substances
9. The components of different batch numbers of this reagent cannot be mixed. |
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