Instruction Manual of Human Osteopontin (OPN) ELISA Kit

Instruction Manual of Human Osteopontin (OPN) ELISA Kit
This kit is only for in vitro research!
Intended application
Quantitative determination of OPN in human serum, plasma, cell culture supernatant or other related biological fluids by ELISA.

Experimental principle
Coat the microwell plate with purified antibody to make a solid-phase carrier. Add specimens or standards, biotinylated anti-OPN antibody, and HRP-labeled avidin to the microwells coated with anti-OPN antibody. After washing, color was 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 color depth is positively correlated with the OPN 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 (lyophilized product): 2 bottles, each bottle is diluted to 1ml with the sample diluent before use, and left to stand for more than 10 minutes after being capped, and then repeatedly inverted / rubbed to help dissolve. / ml, after serial dilutions (Note: do not directly perform multiple dilutions in the plate), dilute them to 10 ng / ml, 5 ng / ml, 2.5 ng / ml, 1.25 ng / ml, 0.625 ng / ml , 0.312 ng / ml, 0.156 ng / ml, the sample dilution is directly used as the standard concentration of 0 ng / ml, prepared within 15 minutes before use. For example, to prepare a 5 ng / ml standard: take 0.5ml (not less than 0.5ml) and add 10 ng / ml of the above standard to an Eppendorf tube containing 0.5ml of sample diluent, mix well, and the rest of the concentration can be deduced by analogy.
3. Sample diluent: 1 × 20ml.
4. Test dilution A: 1 × 10ml.
5. Test dilution B: 1 × 10ml.
6. Detection solution A: 1 × 120μl (1: 100) is diluted with detection diluent A 1: 100 before use, prepared according to the pre-calculated total amount required for each experiment (100μl / well), actual When preparing, 0.1-0.2ml should be prepared. For example, 10μl detection solution A plus 990μl detection dilution A is prepared, mixed gently, and prepared within one hour before use.
7. Test solution B: 1 × 120μl / bottle (1: 100) is diluted 1: 100 with test diluent B before use. The dilution method is the same as that of Test Solution A.
8. Substrate solution: 1 × 10ml / bottle.
9. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop solution: 1 × 10ml / bottle (2N H2SO4).
11. Lamination: 5 sheets
12. Instruction manual: 1 copy

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. Cell culture supernatant or 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.
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.
Sample processing: serum or plasma specimens are recommended to be diluted 10 times, for example: diluted 10 times, 100uL serum or plasma is added to 900uL sample diluent. It is recommended that each laboratory conduct pre-experiment before operation to establish the optimal dilution factor.

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. Add 100μl of sample diluent to blank wells, and add 100μl of standard or test sample to the remaining wells. Be careful not to have air bubbles. Add samples to the bottom of the wells of the microtiter plate. Try not to touch the well walls. The target plate is covered with a cover or film and reacted at 37 ° C for 120 minutes. To ensure the validity of the experimental results, please use a new standard solution for each experiment.
2. Discard the liquid and spin dry without washing. Add 100μl of working solution A (prepared within one hour before use) to each well, add the microplate to the membrane, and react at 37 ℃ for 60 minutes.
3. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, about 400μl / well, spin dry (you can also pat the liquid in the well to pat dry) .
4. Add 100μl of working solution B (the same as the working solution of test A) to each well, and then react with enzyme label plate and film at 37 ℃ for 60 minutes.
5. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 350μl / per well, spin dry (you can also pat the liquid in the well to dry).
6. Add 90μl of substrate solution to each well in sequence, and add the microtiter plate and film to avoid light development at 37 ° C (within 30 minutes, at this time, the first 3-4 wells of the standard product have a clear gradient blue color. 3-4 well gradient is not obvious, you can terminate).
7. Add 50μl of stop solution to each well in sequence to stop the reaction. At this time, 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.
8. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Test immediately 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. Preparation of reagents: Before using A, please shake the hand a few times or centrifuge for a while to prevent the liquid on the tube wall or bottle cap from depositing to the bottom of the tube. Standard products, working solution A, and working solution B should be configured and used according to the required amount, and prepared with the corresponding diluent, not to be confused. Please accurately configure the standard product and working solution, and try not to configure it in a small amount (for example, when drawing the 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, Test solution A working solution or test solution B working solution.
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 OPN at the same time, and has no cross reaction with other related proteins.

Calculation
Taking the concentration of the standard as the vertical coordinate (logarithmic coordinate) and the OD value as the horizontal coordinate (logarithmic coordinate), draw a standard curve on the logarithmic coordinate paper. It is recommended to use professional curve making software for analysis, such as curve expert 1.3, find the corresponding concentration from the standard curve according to the OD value of the sample, and then multiply it by the dilution factor; or use the concentration and OD value of the standard to calculate the regression equation of the standard curve , Substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor, which is the actual concentration of the sample.

Detection range: 0.156 ng / ml-10 ng / ml

Minimum detection limit: 0.078 ng / 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: Short-term (within 1 week) subject to the label, long-term storage Please store all reagents at -20 ℃.
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.

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Instruction Manual of Human Osteopontin (OPN) ELISA Kit

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