Instructions for use of human serum albumin (HSA) ELISA kit Instructions for use of human serum albumin (HSA) ELISA kit
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This kit is only for in vitro research!
Intended application
ELISA method is used to quantitatively determine the content of HSA in human serum, plasma or other related biological fluids.
Experimental principle
This kit uses competitive inhibition enzyme-labeled immunoassay to determine the level of the substance in the specimen. The microtiter plate is coated with the purified antibody to make a solid-phase antibody, and the enzyme-labeled antigen and the test antigen are added to the antibody-coated microwell at the same time, and the test antigen and the enzyme-labeled antigen compete for binding to the specific antibody. After thorough washing, the color is developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The higher the concentration of the specimen to be tested, the more inhibited the binding of the labeled antigen and antibody is, and the lighter the color is. The color depth is positively correlated with the amount of enzyme, but negatively correlated with the content of the substance to be tested in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated.
Kit composition and reagent preparation
1. ELISA plate: one piece (96 wells)
2. Standard product: 2 bottles, each bottle is diluted with 0.8 ml of sample diluent before use. After capping, it is allowed to stand for more than 10 minutes, and then inverted / rubbed repeatedly to help dissolve. Its concentration is 100 ug / mL. After multiple dilutions (Note: Do not directly perform multiple dilutions in the plate), dilute them to 100 ug / mL, 33.3 ug / mL, 11.1 ug / mL, 3.7 ug / mL, 1.23 ug / mL, and the sample dilution directly As a standard concentration of 0 ug / mL, prepare within 15 minutes before use. For example, to prepare a 33.3 ug / mL standard: Take 0.5 ml (not less than 0.5 ml) of the above standard 100 ug / mL and add it to an Eppendorf tube containing 1 ml of sample diluent, mix well. The rest of the concentration can be deduced by analogy.
3. Sample diluent: 1 × 20ml / bottle.
4. Test solution A: 2 × 120ul / bottle (1: 100), diluted with test diluent A 1: 100 before use, prepared according to the pre-calculated total amount required for each experiment before dilution (50ul per well) , The actual preparation should be more 0.1-0.2ml. For example, 1ul detection solution A plus 99ul detection dilution A is prepared in proportion, mix gently and prepare within one hour before use.
5. Test dilution A: 2 × 10ml / bottle.
6. Substrate solution: 1 × 10ml / bottle.
7. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water.
8. Stop solution: 1 × 10ml / bottle (2N H2SO4).
9. Lamination: 1 × 5 sheets
Bring your own items
1. Microplate reader (It is recommended to refer to the instruction manual of the instrument to preheat in advance)
2. Micro pipette and pipette tip, EP tube
3. Distilled or deionized water, new filter paper
Collection and preservation of specimens
1. Serum: Whole blood specimens should be left at room temperature for 2 hours or overnight at 4 ° C and centrifuged at 1000 xg for 20 minutes. Supernatant can be taken for detection, or the specimens should be stored at -20 ° C or -80 ° C, but avoid repeated Freeze and thaw.
2. Plasma: EDTA or heparin can be used as anticoagulant. Centrifuge the sample at 2-8 ° C 1000 xg for 15 minutes within 30 minutes after collection, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing melt.
3. Other biological specimens: centrifuge at 1000 x g for 20 minutes, take the supernatant for testing, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.
4. Sample processing: It is recommended to dilute 500-1,000 times for serum or plasma specimens. Specimens were diluted with 0.1 M PBS (PH = 7.0-7.2).
Note: The above specimens should be stored at 4 ℃ for less than 1 week, -20 ℃ or -80 ℃ should be sealed and stored, -20 ℃ should not exceed 1 month, -80 ℃ should not exceed 2 months; specimen hemolysis will affect the final The test results, so hemolytic specimens should not be tested.
Steps
Before the start of the experiment, all reagents should be equilibrated to room temperature (reagents cannot be dissolved directly at 37 ℃); when the reagents or samples are diluted, they should be mixed evenly. The sample content should be predicted before the experiment. If the sample concentration is too high, the sample should be diluted to make the diluted sample meet the detection range of the kit, and then multiplied by the corresponding dilution factor when calculating.
1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. Dilute the standard and the sample with a centrifuge tube, add 50ul of the standard or the sample to be tested, then immediately add 50ul of the working solution A of the test solution to each well, gently vibrate and mix well, taking care not to have bubbles, add the sample to the enzyme At the bottom of the well of the standard plate, try not to touch the wall of the well, shake gently to mix, add the cover or membrane to the enzyme plate, and react at 37 ℃ for 60 minutes. To ensure the validity of the experimental results, please use a new standard solution for each experiment.
2. Discard the liquid in the hole, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 350ul / per hole, spin dry.
3. Add 90ul of substrate solution to each well in sequence, and develop color at 37 ° C in the dark (within 30 minutes, at this time, the naked eye can see that the standard 3-4 wells have a clear blue gradient, the first 3-4 wells have no obvious gradient , You can terminate).
4. Add 50ul of stop solution to each well in sequence to stop the reaction (in this case, the blue color turns to yellow). The order of adding the stop solution should be the same as that of the substrate solution. In order to ensure the accuracy of the experimental results, the termination solution should be added as soon as possible after the substrate reaction time expires.
5. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Test within 15 minutes after adding stop solution.
Note:
1. Reagent preparation: All reagents must reach room temperature before use. Please save the reagents according to the instructions immediately after use. Please use disposable tips during the experiment to avoid cross contamination.
2. Add sample: When adding samples or reagents, please note that when drawing samples / standards, enzyme conjugates or substrates, if the time interval between the first well and the last well is too large, it will be This results in different "pre-incubation" times, which obviously affect the accuracy and repeatability of the measured values. It is best to control the time of one sample (including the standard and all samples) within 10 minutes. If the number of samples is large, it is recommended to use a multi-channel pipette.
3. Incubation: To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test, and add a cover or film to the enzyme label plate to avoid liquid evaporation; the next step should be carried out as soon as possible after washing Whenever possible, the enzyme plate should be kept in a dry state; at the same time, the given incubation time and temperature should be strictly observed.
4. Washing: The washing liquid remaining in the reaction well during the washing process should be fully patted dry on the filter paper. Do not put the filter paper directly into the reaction well to absorb water. At the same time, the remaining liquid and fingerprints on the bottom of the plate should be eliminated to avoid affecting the final enzyme. Calibrator readings.
5. Reagent preparation: Before using A, please shake your hands a few times or centrifuge for a short time, so that the liquid on the tube wall or bottle cap is deposited on the bottom of the tube. The standard product and test solution A working fluid should be configured and used according to the required amount, and prepared with the corresponding diluent, not to be confused. Please configure the standard product and working solution accurately, and try not to dispense it in a small amount (for example, when drawing test solution A, it should not be less than 10μl at a time) to avoid the concentration error caused by inaccurate dilution; do not reuse the diluted standard product or Test solution A working fluid.
6. Control of reaction time: after adding the substrate, please observe the color change of the reaction well regularly (for example, every 10 minutes). If the color is darker, please add the stop solution in advance to stop the reaction to avoid the reaction being too strong and affecting the microplate reader Optical density reading.
7. Substrate: Please keep the substrate away from light, and avoid direct exposure to strong light during storage and incubation.
It is recommended to set a double-hole test when testing samples to ensure the accuracy of the test results.
If the content of the substance to be tested in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor.
Washing method
1. Manual plate washing method: aspirate (do not touch the plate wall) or shake off the liquid in the microplate; place a few layers of absorbent paper on the experimental table, and pat the microplate down several times with force; apply the recommended washing buffer Inject at least 0.3ml into the hole, soak for 1-2 minutes, and repeat this process several times as needed.
2. Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.
Specificity
The kit can detect recombinant or natural human HSA at the same time, and has no cross reaction with other related proteins.
Calculation
Taking the concentration of the standard as the ordinate (logarithmic coordinate) and the OD value as the abscissa, draw a standard curve on the logarithmic coordinate paper. According to the OD value of the sample, find the corresponding concentration from the standard curve and multiply it by the dilution factor; or calculate the regression equation of the standard curve using the concentration of the standard and the OD value, and substitute the OD value of the sample into the equation to calculate the sample concentration. Multiply by the dilution factor to get the actual concentration of the sample.
Detection range: 1.23 ug / mL-100 ug / mL
Minimum detection limit: 0.3 ug / mL
Explanation
1. Avoid exposing the reagent to strong light during storage and incubation. All reagent bottle caps must be tightly closed to prevent evaporation and contamination. Reagents should be protected from microbial contamination, because the interference of proteolytic enzymes will lead to erroneous results.
2. Storage of the kit: Please store the standard, detection solution A and detection solution B at -20 ℃ as soon as possible after receiving the kit. The other reagents should be stored at 4 ℃ for short-term storage and -20 ℃ for long-term storage.
3. Salt will be precipitated from the concentrated washing liquid, which can be heated and dissolved in the water bath when diluted.
4. There may be a little water-like substance in the well of the enzyme-linked plate just opened. This is normal and will not have any impact on the experimental results.
5. All samples should be well managed, and the samples and testing devices should be processed according to the prescribed procedures.
6. Validity: 6 months.
7. These operating instructions apply to the 48T kit, but all reagents in the 48T kit are halved.
Xiamen Huijia Biotechnology Co., Ltd. is a professional agent for many well-known brands in the international life science field. Committed to the sales and promotion of various ELISA kits, immunohistochemical kits, primary and secondary antibodies, cytokines, biological reagents, pipettes, and consumables.
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