Physical properties
Each liquid component is clear and transparent, without precipitation or floccules. Microplate aluminum foil bagIt should be vacuum packed without damage or leakage.
Use
Used to detect the concentration of human Rh blood type D antigen (RHD) in samples such as serum, plasma, and cell culture supernatant.
Experimental principle
This kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA). In a microwell ELISA plate pre-coated with anti-human Rh blood type D antigen (RHD) antibody (solid phase antibody), add human Rh blood type D antigen (RHD) calibrator and the sample to be tested, then add biotin-labeled antibody, incubate and fully wash, then add HRP-coupled avidin, incubate and fully wash to remove unbound components, and form a sandwich complex of solid phase antibody-antigen-biotin-labeled antibody-avidin enzyme on the solid surface of the microwell plate. Add TMB colorimetric solution to produce a blue product, which eventually turns into yellow under the action of the stop solution. The absorbance (OD value) is measured at a wavelength of 450nm on an ELISA instrument. The absorbance (OD value) is positively correlated with the concentration of human Rh blood type D antigen (RHD) in the sample to be tested. Fitting the calibrator curve can calculate the concentration of human Rh blood type D antigen (RHD) in the sample.
Specificity
This kit recognizes natural human Rh blood group D antigen (RHD) and has no cross-reactivity with structural analogs.
Repeatability
The coefficient of variation within and between plates was<10%.
Recovery
The recovery rate is 85%-115%between.
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
Recommended sample dilution plan: It is recommended that teachers conduct preliminary experiments to explore the optimal dilution multiple of the sample before conducting formal experiments.All reagents and components should be returned to room temperature first. For calibrators, quality control products, and samples, duplicate wells are recommended. 12. Prepare the working solutions of various components of the kit according to the method described in the previous instructions. 2, 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. 3, set up calibration wells, sample diluent wells, blank wells and sample wells, and add different concentrations of calibration wells to each calibration well50μL, add sample diluent to the sample diluent well50μL, no addition is made to the blank wells, and the sample wells are added with the sample to be tested50μLBiotin antibody was added to each well except the blank well.100uL, cover the reaction plate with a sealing film,37Incubate in a water bath or incubator away from light60min. 4, remove the sealing film, discard the liquid, pat dry on absorbent paper, fill each well with washing solution, and let it stand1min, shake off the detergent, pat dry on absorbent paper, and repeat this process.5If you use an automatic plate washer, please follow the plate washer operating procedures to wash the plate and add soaking30sAfter washing the plate, pat the plate dry on a clean, lint-free paper before adding the substrate. 5, except for the blank well, addHRPLabeled avidin100uL, cover the reaction plate with a sealing film,37Incubate in a water bath or incubator away from light20min. 6, repeat steps4. 7, add to all wellsTMBColor development solution100μL. Cover the reaction plate with a sealing film.37Incubate in a water bath or incubator away from light15min. 8, add stop solution to all wells50μL,exist450nmRead the absorbance of each well on a wavelength microplate reader (ODvalue).
Announcements
1, This kit is for in vitro research use only and is not 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, incubate strictly according to the specified time and temperature to ensure accurate results. All reagents must reach room temperature before use 20-25℃. Refrigerate the reagent immediately after use. 4Improper plate washing may lead to inaccurate results. Make sure to aspirate as much liquid as possible before adding substrate. Do not allow the wells to dry out during incubation. 5, remove the residual liquid and fingerprints on the bottom of the board, otherwise it will affect ODvalue. 6, the substrate developing solution should be colorless or very light in color. 7, avoid cross contamination of reagents and specimens to avoid erroneous results. 8, Avoid direct exposure to strong light during storage and incubation. 9equilibrate to room temperature before opening the sealed bag to prevent water droplets from condensing on the cold strips. 10, Any reaction reagent cannot come into contact with bleaching solvent or the strong gas emitted by bleaching solvent. Any bleaching component will destroy the biological activity of the reaction reagent in the kit. 11, the ELISA reader used for testing needs to be installed with the ability to detect 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 replace the reagents in this kit with reagents from other batches or other sources. 13, used in the test 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: The positive and negative control results are unstablePossible 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 temperature 3.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.
Statement
The kit is for research use only. If it is used for clinical diagnosis or any other purpose, our company will not be responsible for any problems arising therefrom and will not bear any legal liability.
