The usage method of the ApoC2 assay kit may vary depending on different brands and models, but generally speaking, the steps for its use are roughly the same. Here is a universal usage method based on multiple brands of reagent kits for your reference:
1、 Preparation stage
Take out the reagent kit: Take out the reagent kit from the refrigerator in advance and let it equilibrate to room temperature (usually taking 10-20 minutes).
Preparation of standard samples: According to the instructions, add freeze-dried standard samples to a universal diluent, let them dissolve, gently mix them, and dilute them by multiple ratios to obtain a series of standard working solutions with different concentrations.
Preparation of working solution: According to the instructions, prepare biotinylated antibody working solution, enzyme conjugate working solution, and washing solution.
2、 Operation steps
Take out Flat noodles: take out the required number of Flat noodles from the aluminum foil bag, and seal the remaining Flat noodles with a self sealing bag and put them back into the refrigerator for storage.
Set the hole position: set the standard hole, sample hole and blank hole on the Flat noodles. Add different concentrations of standard working solution to the standard well, add the test sample to the sample well, and add universal diluent to the blank well.
Sample addition: According to the instructions, add an appropriate amount of sample or standard working solution to each well, usually 100 μ L per well. After adding the sample, seal the reaction hole with a sealing film.
Warming: put the Flat noodles into a 37 ℃ incubator and warm it in dark for a certain time (such as 60 minutes).
Washing: Discard the liquid in the hole, and wash the Flat noodles repeatedly with the washing liquid. Usually, it needs to be washed 3~5 times. After each washing, pat it on the absorbent paper.
Enzyme conjugate addition: Add an appropriate amount of enzyme conjugate working solution to each well, seal the reaction well again, and incubate in a 37 ℃ constant temperature incubator in the dark for a certain period of time (such as 30 minutes).
Re washing: wash the Flat noodles again according to the method in step 5.
Add substrate: Add an appropriate amount of substrate solution (such as TMB) to each well, seal the reaction well, and incubate in a 37 ℃ constant temperature incubator in the dark for a certain period of time (such as 15 minutes).
Add stop solution: take out the Flat noodles, add proper stop solution to each hole, and stop the reaction.
Measure OD value: Measure the OD value of each well using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450nm.
3、 Result calculation
Calculate correction value: Calculate the average OD value of the standard and sample wells, and subtract the OD value of the blank well as the correction value.
Draw standard curve: Plot the standard curve of the four parameter logic function on a double logarithmic coordinate paper with the concentration of the standard substance as the horizontal axis and the OD value as the vertical axis.
Calculate sample concentration: Based on the OD value of the sample, search for the corresponding concentration value on the standard curve. If the OD value of the sample is higher than the upper limit of the standard curve, it should be diluted appropriately and retested, and multiplied by the corresponding dilution factor when calculating the sample concentration.
4、 Precautions
Strictly follow the instructions: Different brands and models of reagent kits may have different operating requirements and precautions. Please read the instructions carefully and operate strictly according to the requirements.
Avoid cross contamination: When using a micropipette, the suction tips should not be mixed to prevent cross contamination.
Control reaction time and temperature: Strictly control the time and temperature of each reaction step to ensure the accuracy of the results.
Timely reading of results: After adding the termination solution, the results should be read on the enzyme-linked immunosorbent assay reader in a timely manner to avoid inaccurate results due to prolonged time.
Storage and disposal: The reagent kit should be stored under specified temperature conditions, avoiding direct sunlight and moisture. The used reagent kits and samples should be disposed of in accordance with local government and national regulations.
Please note that the above steps and precautions are for reference only. Specific use should be carried out according to the instructions of the reagent kit.
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