| Usage | Specimen requirements:
1. Specimen collection is extracted as soon as possible, after extraction, according to the literature should be as soon as possible after extraction experiments. If not immediately, can put in the specimen - 20 ℃ to save, but should avoid repeated freezing and thawing
2. Can't test samples containing NaN3, by NaN3 inhibition of horseradish peroxidase (HRP) activity.
steps:
1. The standard dilution: this kit provides the original times standard, the user can according to the following chart in small tube dilution.
| 320 ng/mL | Standard 5 | 150uL original standard added 150uL standard dilution | | 160ng/mL | Standard 4 | 150uL standard 5 add 150uL standard dilution | | 80ng/mL | Standard 3 | 150uL standard 4 add 150uL standard dilution | | 40ng/mL | Standard 2 | 150uL standard 3 add 150uL standard dilution | | 20ng/mL | Standard 1 | 150uL of standard No. 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: each Kong Xian join A 50 ul agent, adding chromogenic agent B 50 ul, gently shake blending, 37 ℃ avoid light color 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 with the concentration of the standard content of standard curve and the OD value calculated linear regression equation, the sample OD value into the equation, to calculate the concentration of sample, then multiplied by the dilution ratio, which is the actual concentration of the sample. |
| General Notes | 1. The kit should be equilibrated at room temperature for 1 hour before use when removed from the refrigerated environment. If the enzyme-labeled coated plate is not used up after opening, the slat should be stored in a sealed bag.
2. Crystallization may be precipitated in the concentrated washing solution, which can be heated in a water bath when diluted, and does not affect the results when washing.
3. A sampler should be used for each step of sample addition, and its accuracy should be checked frequently to avoid test errors. It is best to control the sample adding time within 5 minutes. If the number of samples is large, it is recommended to use the gun adding sample.
4. Please make a standard curve at the same time of each measurement, and it is better to make 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 sample), please dilute a certain number (n times) with the sample diluent before determination, and multiply by the total dilution (× n× 5).
5. The sealing plate film is only used once to avoid cross contamination.
6. The substrate should be kept away from light.
7. The operation was carried out in strict accordance with the instructions, and the test results must be judged by the reading of the microplate reader.
8 All samples, washes and waste of all kinds should be treated as infectious agents.
9. This reagent for different batch number components shall not be mixed. |
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