Operation steps 1. Dilution of standard product: This kit provides one original standard product. Users can dilute it in a small test tube according to the following chart. 20ng/ml | Standard No. 5 | 150μl of primary standard was added to 150μl of standard dilution | 10ng/ml | Standard No. 4 | 150μl of standard No. 5 was added to 150μl of standard dilution | 5ng/ml | Standard No.3 | 150μl of standard No. 4 was added to 150μl of standard dilution | 2.5 ng/ml | Standard No.2 | 150μl of standard No. 3 was added to 150μl of standard dilution | 1.25 ng/ml | Standard No. 1 | 150μl of standard No. 2 was added to 150μl of standard dilution |
2. Sample addition: Set up blank wells (the blank control wells do not add samples and enzyme-labeled reagents, and the other steps are the same), standard wells, and sample wells to be tested respectively. Accurately add 50μl of the standard on the enzyme-labeled coated plate, first add 40μl of sample diluent to the sample well to be tested, and then add 10μl of the sample to be tested (the final dilution of the sample is 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: Incubate at 37 °C for 30 minutes after sealing with a plate sealing film. 4. Liquid preparation: 30 times concentrated washing solution is diluted 30 times with distilled water and used for later use 5. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each hole with washing liquid, let it stand for 30 seconds and then discard it. Repeat this 5 times and pat dry. 6. Enzyme addition: 50μl of enzyme-labeled reagent was added to each well, except for blank wells. 7. Incubation: The procedure is the same as 3. 8. Washing: The operation is the same as 5. 9. Color development: Add 50μl of color developer A50 μl to each well, then add 50μl of color developer B50 μl, gently shake and mix well, and develop color at 37 °C in the dark for 10 minutes. 10. Termination: Add 50μl of stop solution to each well to terminate the reaction (blue immediately turns yellow at this time). 11. Determination: Zero the blank hole, and measure the absorbance (OD value) of each hole sequentially at a wavelength of 450nm. The determination should be performed within 15 minutes after the addition of the stop solution.
calculate Taking the concentration of the standard substance as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, and find out the corresponding concentration from the standard curve according to the OD value of the sample; Multiply by the dilution factor; Or use the concentration of the standard substance and the 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 it by the dilution factor, which is the actual concentration of the sample. | 
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