Test equipment required for the test
1, standard specification ELISA reader. 2automatic plate washer. 3, oscillator. 4, series of adjustable pipettes and tips. When testing a large number of samples at one time, it is best to use a multi-channel pipette.
Running program
1, all reagents and components should be restored to room temperature first. It is recommended to perform duplicate wells for standards, quality control products and samples. 22. Prepare the working solutions of various components of the kit according to the method described in the previous reagent preparation. 3, take out the required strips from the aluminum foil bag, seal the remaining strips in a ziplock bag and put them back in the refrigerator. 4, set up standard wells, sample wells and blank wells, and add different concentrations of standards to each standard well50μL, add the sample to be tested to the sample well50μL, do not add any blank wells, and cover the reaction plate with a sealing film.37℃ water bath or incubator30min. 5, remove the sealing film, discard the liquid, pat dry on absorbent paper, fill each well with washing solution, and let it stand20s, shake off the detergent and pat dry on absorbent paper. Repeat this process.4times (total wash plate5If using an automatic plate washer, follow the plate washer operating procedures to wash the plate and add soaking20sAfter washing the plate, pat the plate dry on a clean, lint-free paper before adding the substrate. 6, In addition to the blank wells, the standard wells and sample wells, add horseradish peroxidase-labeled detection antibodies100μL. 7, Cover the reaction plate with sealing film,37℃ water bath or incubator30min. 8, remove the sealing film, discard the liquid, pat dry on absorbent paper, fill each well with washing solution, and let it stand20s, shake off the detergent and pat dry on absorbent paper. Repeat this process.4times (total wash plate5If using an automatic plate washer, follow the plate washer operating procedures to wash the plate and add soaking20sAfter washing the plate, pat the plate dry on a clean, lint-free paper before adding the substrate. 9, the substrateAandBaccording to1:1Mix the volume thoroughly and add the substrate mixture to all wells.100μL. Cover the reaction plate with a sealing film.37℃ water bath or incubator15min. 10, add stop solution to all wells50μL, in the microplate reader450nmRead the absorbance of each well at wavelength (ODvalue).Result calculation 11, with the concentration of the standard as the horizontal axis, the corresponding absorbance (ODvalue) as the ordinate, using computer software, using four parametersLogisticCurve fitting (4-pl), create a standard curve equation, through the absorbance of the sample (ODvalue), and use the equation to calculate the concentration of the sample.ELISA CalcSoftware calculation】 12If the sample is diluted, the concentration value measured by the above method must be multiplied by the dilution factor to obtain the final concentration of the sample.
Announcements
1)This kit is for in vitro research use only and is not intended for clinical diagnosis. 2)Please wear lab coats and latex gloves for protection during the test. Especially when testing blood or other body fluid samples, please follow the national biological laboratory safety protection regulations. 3)Strictly follow the specified time and temperature to incubate to ensure accurate results. All reagents must reach room temperature before use. 20-25℃. Refrigerate the reagent immediately after use. 4)Improper plate washing can lead to inaccurate results. Be sure to aspirate as much liquid as possible before adding substrate. Do not allow the wells to dry out during incubation. 5)Eliminate residual liquid and fingerprints on the bottom of the board, otherwise it will affect ODvalue. 6)The substrate developer solution should be colorless or very light in color. 7)Avoid cross contamination of reagents and specimens to avoid erroneous results. 8)Avoid direct sunlight during storage and incubation. 9)The sealed bag was opened after equilibration to room temperature to prevent water droplets from condensing on the cold strips. 10)Any reagent should not be exposed to bleaching solvents or the strong gases emitted by bleaching solvents. Any bleaching components will destroy the biological activity of the reagents in the kit. 11)The ELISA reader used for the test needs to be installed with 450±10nmWavelength filters with optical density range of 0-3.5It is recommended to use in advance 15Minutes to preheat. 12)Do not mix or substitute reagents in this kit with reagents from other lots or other sources. 13)The test used EPBoth the tube and the pipette tip are for single use and mixing is strictly prohibited. 14)Do not use expired reagents.
Problem analysis
①Problem description: Standard curve gradient is poorPossible causes and corresponding countermeasures: 1.Inaccurate aspiration or addition of liquid——Check the pipette and tips 2.Equilibration time is too short——Ensure adequate balancing time 3.Incomplete washing——Ensure the washing time, washing times and liquid volume per well ②Problem description: The color is very weak or colorlessPossible causes and corresponding countermeasures: 1.Incubation time is too short——Ensure adequate incubation time 2.Incorrect test temperature——Use the recommended test temperature3.Insufficient reagent volume or missed addition/Incorrect dilution——Check the aspiration and addition process to ensure that all reagents are added in order and in sufficient quantities. 4.Enzyme marker inactivation or substrate failure——Mix the enzyme conjugate and substrate and check and judge by rapid color development ③Problem description: Low readingsPossible causes and corresponding countermeasures: 1.The microplate reader is not set up correctly——Check wavelength and filter settings on the microplate reader/Turn on the ELISA reader in advance to preheat ④Problem description: Large coefficient of variationPossible causes and corresponding countermeasures: 1.Incorrect addition of liquid——Check the filling status ⑤Problem description: High background valuePossible causes and corresponding countermeasures: 1.The working concentration of the detection antibody is too high——Use the recommended dilution factor 2.Incomplete washing of ELISA plate——Ensure that each step is cleaned completely; if using an automatic plate washer, check whether all outlets are blocked; whether the washing solution provided in the kit is used 3.The washing liquid is contaminated——Prepare new lotion ⑥Problem description: Low sensitivityPossible causes and corresponding countermeasures: 1.ELISAImproper storage of the test kit——Store relevant reagents according to the instructions 2.Not terminated before reading——ODStop solution should be added to each well before reading.
