The precautions for using the anti trypsin assay kit are as follows:
Sample processing and storage:
Serum samples: Use test tubes without pyrogens and endotoxins to avoid cell irritation. After collecting blood, centrifuge at 1000 × g for 10-20 minutes to separate the serum and avoid hemolysis and hyperlipidemia. If there are particles in the serum, centrifugation or filtration is required.
Plasma samples: EDTA, citrate or heparin are used as anticoagulants. Within 30 minutes after collection, centrifuge at 1000 × g for 15-30 minutes at 2-8 ℃, and collect the supernatant.
. Cell supernatant: Centrifuge at 1000 × g for 10-20 minutes to remove particles and polymers.Tissue homogenate: Wash the tissue with pre cooled PBS (0.01M, pH=7.4), remove residual blood, weigh and cut into pieces, add PBS (protease inhibitor can be added) at a ratio of 1:9 (weight to volume), grind thoroughly on ice or sonicate, centrifuge at 5000 × g for 5-10 minutes, and take the supernatant.
. Storage conditions: If the sample is not used immediately, it should be packaged and stored at -70 ℃ to avoid repeated freezing and thawing. Thawing should be carried out at room temperature to ensure that the sample is evenly and fully thawed.Preparation and use of reagents:
Storage of reagent kit: The reagent kit should be stored at 2-8 ℃ and equilibrated at room temperature for 20-30 minutes before use.
. Wash buffer dilution: Dilute with distilled water according to the instructions (such as 1:20 or 1:50) to ensure even dilution.Standard dilution: Dilute the series according to the instructions, use and prepare immediately, do not store.
. Biotin labeled antibodies and affinity chain enzyme HRP: usually ready to use, can be used directly.Avoid cross contamination: Try not to touch the hole wall when adding samples, gently shake and mix well.
. If the sample concentration is too high, it needs to be diluted with sample diluent before adding the sample, and the calculation should be multiplied by the corresponding dilution factor.Experimental operation specifications:
Sample addition sequence and quantity: Strictly follow the time, sample addition quantity, and sequence indicated in the instructions for operation.
. Add 50 μ L of standard samples of different concentrations to each standard well, add 50 μ L of the test sample to the sample well, and do not add blank wells.Incubation conditions: In addition to blank wells, 100 μ L of horseradish peroxidase (HRP) labeled detection antibody is added to each well of the standard and sample wells. The reaction well is sealed with a sealing membrane and incubated at 37 ℃ in a water bath or constant temperature incubator for 60 minutes (the specific time is adjusted according to the instructions of the kit).
. Ensure that all reagents reach room temperature (20-25 ℃) before use, and do not let the micropores dry out during the incubation process. Washing steps: Discard the liquid and 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, and repeat washing the board 5 times (or use a washing machine to wash the board). Insufficient washing can lead to accuracy errors and incorrect increase in OD values.Color development and termination: 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).
. The substrate color solution should be colorless or very light in color, and the substrate solution that has turned blue cannot be used. Add 50 μ L of termination solution to each well, and measure the OD value of each well at 450nm wavelength within 15 minutes (specific time adjusted according to the kit instructions). The order of adding termination solution should be as similar as possible to the order of adding substrate solution.Result determination and calculation:
OD value determination: Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be carried out immediately after adding the stop solution.
. Standard curve drawing: 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 using relevant software, and obtain a linear regression equation. Sample concentration calculation: Substitute the OD value of the sample into the equation to calculate the concentration of the sample. If the sample has been diluted, it needs to be multiplied by the corresponding dilution factor to obtain the actual concentration.Other precautions:
Avoid direct sunlight: Avoid exposing the reagents to strong light during storage and incubation.
.Preventing reagent evaporation and contamination: All reagent bottle caps must be tightly closed to prevent evaporation and contamination. Reagents should avoid contamination by microorganisms, as interference from proteolytic enzymes can lead to incorrect results.
.Reagent expiration date: Pay attention to the expiration date of the reagent kit, expired products cannot be used.
. Biosafety: Testing must comply with laboratory management regulations and strictly prevent cross contamination. All samples, washing liquids, and various waste materials should be disposed of as infectious agents. The liquid components of the reagent kit may contain preservatives, which may cause skin allergic reactions. Avoid inhaling smoke, wear protective gloves during operation, and wash hands thoroughly after the experiment is completed.source:http://en.zjikon.com/news1082017.html