The usage method of the antithrombin III assay kit (using enzyme-linked immunosorbent assay (ELISA) as an example) is as follows:
1. Preparation before the experiment
Preparation of reagents and equipment:
Ensure that you have the necessary equipment such as an enzyme-linked immunosorbent assay (ELISA) reader (450nm wavelength), high-precision sampler and nozzle (0.5-10 μ L, 2-20 μ L, 20-200 μ L, 200-1000 μ L), 37 ℃ constant temperature box, distilled water or deionized water.
Check the composition of the reagent kit, including standard samples, enzyme-linked immunosorbent assay (ELISA) coated plates, ELISA reagents, sample diluents, color reagents A and B, stop buffer, concentrated washing solution, etc., to ensure that all reagents are within their expiration date and the storage conditions meet the requirements.
.Sample preparation:
Serum sample: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or overnight at 4 ℃, then centrifuge at 1000 × g for 20 minutes, and take the supernatant.
. Alternatively, store the supernatant at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.Plasma samples: Collect samples using EDTA or heparin as anticoagulants, and centrifuge the samples at 2-8 ℃ 1000 × g for 15 minutes within 30 minutes after collection. The supernatant can be collected for detection.
. Alternatively, store the supernatant at -20 ℃ or -80 ℃ to avoid repeated freezing and thawing. Tissue homogenate sample: Rinse the tissue with pre cooled PBS (0.01M, pH=7.4) to remove residual blood. After weighing, cut the tissue into pieces and add it to a glass homogenizer with the corresponding volume of PBS (usually in a weight to volume ratio of 1:9). Grind thoroughly on ice. To further lyse tissue cells, the homogenate can be subjected to ultrasonic fragmentation or repeated freeze-thaw cycles. Finally, centrifuge the homogenate at 5000 × g for 5-10 minutes and collect the supernatant for detection.Cell culture supernatant or other biological specimens: please centrifuge at 1000 × g for 20 minutes, and take the supernatant for detection.
. Or store the supernatant at -20 ℃ or -80 ℃ to avoid repeated freezing and thawing.Reagent reheating:
Remove the reagent kit from the refrigerated environment and allow it to equilibrate at room temperature for 20-30 minutes before use.
.Dilute the concentrated washing solution at a ratio of 1:20, which is 1 part of 20 x washing buffer and 19 parts of distilled water.
.II. Experimental operation steps
Sample adding:
Remove the required Flat noodles from the aluminum foil bag after room temperature equilibrium, and seal the remaining Flat noodles with self sealing bags and put them back at 4 ℃.
Set standard wells and sample wells, add 50 μ L of different concentrations of standard to each standard well;
; Add 50 μ L of the test sample to the sample well; Blank holes are not added. Incubation: Except for blank wells, 100 μ L of horseradish peroxidase (HRP) labeled detection antibody was added to each well of the standard and sample wells. Seal the reaction well with a sealing film and incubate at 37 ℃ in a water bath or constant temperature box for 60 minutes (the specific time should be adjusted according to the instructions of the reagent kit).Washing:
Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μ L), let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, repeat washing the board 5 times (or use a washing machine to wash the board).
.Color development:
Add 50 μ L of substrate A and 50 μ L of substrate B to each well, and incubate at 37 ℃ in the dark for 15 minutes (the specific time should be adjusted according to the instructions of the kit).
.Termination:
Add 50 μ L of termination solution to each well to terminate the reaction (at this point, the blue color turns yellow).
.Measurement:
Measure the OD values of each well at a wavelength of 450nm within 15 minutes.
.III. Result Calculation and Judgment
Draw a standard curve:
Using the OD value of the measured standard as the horizontal axis and the concentration value of the standard as the vertical axis, draw the standard curve on a coordinate paper or with relevant software, and obtain a linear regression equation.
. Calculate sample concentration: Substitute the OD value of the sample into the linear regression equation to calculate the concentration of the sample. If the sample concentration exceeds the detection range of the standard curve, it is necessary to dilute the specimen appropriately with sample diluent before conducting the experiment, and multiply the calculation by the total dilution factor.IV. Precautions
Operating standards:
Strictly follow the instructions of the reagent kit and the experimental operation steps to avoid operational errors that may lead to inaccurate experimental results.
.Sample dispensers should be used for each step of sample addition, and their accuracy should be regularly 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 sampling gun for sample addition.Reagent storage:
All reagents must reach room temperature of 20-25 ℃ before use.
. Immediately refrigerate and store the reagents after use to avoid reagent failure. Different batches of reagents must not be mixed. Washing and color development: Incorrect washing can lead to inaccurate results. Ensure to absorb as much liquid as possible from the well before adding the substrate. Do not let the micropores dry out during the incubation process. The substrate color solution should be colorless or very light in color, and the substrate solution that has turned blue cannot be used.Avoid contamination:
Avoid cross contamination between reagents and specimens to prevent erroneous results.
. The sealing film is only for one-time use to avoid cross contamination.Safety protection:
During the experiment, it is necessary to wear experimental clothing and disposable gloves to ensure personal safety.
. All samples, detergents, and various waste materials should be treated as infectious agents to avoid environmental pollution.source:http://en.zjikon.com/news1082477.html