User Manual of Human Formyl Methionine (fMet) ELISA Kit

This kit is only for in vitro research!
Intended application
Quantitative determination of fMet in human urine or other related biological fluids by ELISA.

Experimental principle The microtiter plate is coated with purified antibody to make a solid phase carrier, and the specimen or standard, biotinylated anti-fMet antibody, and HRP-labeled avidin are sequentially added to the microwell coated with anti-fMet 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 color depth is positively correlated with fMet 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 (freeze-dried product): 2 bottles, each bottle is diluted with sample diluent to 1ml before use, and left to stand for more than 10 minutes after being capped, and then inverted / rubbed repeatedly to help dissolve, its concentration is 100 ug / ml, after serial dilutions (Note: do not directly perform multiple dilutions in the plate), dilute them to 100 ug / ml, 50 ug / ml, 25 ug / ml, 12.5 ug / ml, 6.25 ug / ml , 3.12 ug / ml, 1.56 ug / ml, the sample dilution is directly used as the standard concentration of 0 ug / ml, prepared within 15 minutes before use. For example, to prepare a 50 ug / ml standard: take 0.5ml (not less than 0.5ml) and add 100 ug / 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 before the dilution 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. Detection solution B: 1 × 120μl / bottle (1: 100) is diluted 1: 100 with detection 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. Urine: Please collect the first urine in the morning (mid-stage urine), or 24-hour urine, collect the supernatant after centrifugation at 2000 xg for 15 minutes, and store the specimen at -20 ℃, and avoid repeated freezing and thawing.
2. Other biological specimens: centrifuge at 1000 xg 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;

Before starting the experiment, all reagents should be equilibrated to room temperature (reagents can not be dissolved directly at 37 ℃); when the reagents or samples are diluted, they should be mixed well, and try to avoid foaming when mixing. 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 the working solution of detection solution A (prepared within one hour before use) to each well, add the microplate to the membrane, and react at 37 ° C 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 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 three to four wells of the standard product have a clear gradient blue color after the naked eye. 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 specimens 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 the plate. 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 residual liquid and fingerprint 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 test solution A, it should not be less than 10μl at a time) to avoid the concentration error caused by inaccurate dilution; Test solution A working solution or test solution B working solution.
6. Control of reaction time: Please observe the color change of the reaction well regularly after adding the substrate (for example, every 10 minutes). If the color is dark, 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; place the recommended washing buffer Inject at least 0.3ml into the hole and soak for 1-2 minutes. 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 This kit can detect recombinant or natural human fMet at the same time, and does not cross-react with other related proteins.

Calculation The concentration of the standard is taken as the ordinate (logarithmic coordinate), the OD value is the abscissa (logarithmic coordinate), and a standard curve is drawn 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.

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. Aspirate the reagents carefully and strictly observe the given incubation time and temperature. 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 result in different "pre-incubation" time, which obviously Affect the accuracy and repeatability of the measured value. Moreover, insufficient washing will affect the test results.
3. Storage of the kit: some reagents are stored at -20 ° C, and some reagents are stored at 2-8 ° C, depending on the label.
4. Salt will precipitate out in the concentrated washing liquid, and it can be heated and dissolved in the water bath when diluted.
5. There may be some water-like substances in the well of the enzyme-linked plate just opened. This is a normal phenomenon and will not have any impact on the experimental results.
6. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.
7. All samples should be managed, and the samples and testing devices should be processed according to the prescribed procedures.
8. Validity: 6 months

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