Instruction manual of ELISA kit for specific marker of human bone degeneration (CTX-2)

This kit is for in vitro research use only

Intended application

Quantitative determination of bone degradation specific marker (CTX-2) in human serum, plasma, cell culture supernatant 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-CTX-2 antibody, and HRP labeled affinity are added to the microwell coated with anti-CTX-2 antibody in sequence. After thorough washing, it 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 CTX-2 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 of which is diluted with sample diluent to 1ml before use. After being capped, it is allowed to stand for more than 10 minutes, and then repeatedly inverted / rubbed to help dissolve. Its concentration is 5,000 pg / ml, after serial dilution, separate dilution

5,000 pg / ml, 2,500 pg / ml, 1,250 pg / ml, 625 pg / ml, 312 pg / ml, 156 pg / ml, 78 pg / ml Prepare within 15 minutes.

To prepare 2,500 pg / ml standard: Take 0.5ml (not less than 0.5ml) of the above standard at 5,000 pg / ml and add it to an Eppendorf tube containing 0.5ml of sample diluent, mix well. The rest of the concentration can be deduced by analogy.

3. Sample diluent: 1 × 20ml / bottle.

4. Test the diluent A: 1 × 10ml / bottle.

5. Test diluent B: 1 × 10ml / bottle.

6. Detection solution A: 1 × 120ul / bottle (1: 100), diluted with detection dilution A 1: 100 before use, prepared according to the pre-calculated total amount required for each experiment before dilution (100ul per well) , The actual preparation should be more 0.1-0.2ml. For example, 10 ul detection solution A plus 990 ul detection dilution A is prepared, mix gently, and prepare within one hour before use.

7. Test solution B: 1 × 120ul / 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).

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 ° C or -80 ° C, but avoid repeated freezing melt.

3. Cell culture supernatant or other biological specimens: centrifuge at 1000 xg for 20 minutes, take the supernatant for detection, 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, -20 ℃ should not exceed 3 months, -80 ℃ should not exceed 6 months; specimen hemolysis will affect the final As a result of the test, hemolytic specimens should not be tested; specimens with high blood lipids do not require special treatment and can be tested directly.

Steps

Before the experiment, please prepare all the reagents in advance; when the reagents or samples are diluted, they should be mixed well, and try to avoid foaming when mixing. A standard curve should be made for each test. If the sample concentration is too high, the sample diluent should be used for dilution to make the sample meet the detection range of the kit. It is recommended that each laboratory conduct pre-experiment before operation to establish the optimal dilution factor.

1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. Add 100ul of sample diluent to the blank well, and 100ul of the standard or the sample to be tested in the remaining well. Be careful not to have air bubbles. Add the sample to the bottom of the well of the microplate. 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 100ul of detection solution A working solution (prepared within one hour before use) to each well, 37 ℃, 60 minutes.

3. After incubating for 60 minutes, discard the liquid in the hole, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, 350ul / per hole, spin dry (you can also pat the liquid in the hole to pat dry).

4. Add 100 μl of testing solution B working solution (same as testing A working solution) to each well at 37 ℃ for 60 minutes.

5. After incubating for 60 minutes, 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 (you can also pat the liquid in the hole to pat dry).

6. 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 first 3-4 wells of the standard product have a visible blue gradient, and the latter 3-4 wells have no obvious gradient) , You can terminate).

7. Add 50ul 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. Leave one hole for each experiment as a blank zero adjustment hole (different from the blank hole). No reagents are added to this hole, but the substrate solution and 2N H2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring.

2. Incubate in strict accordance with the prescribed time and temperature to ensure accurate results. All reagents must reach room temperature before use. Refrigerate the reagent immediately after use.

3. Incorrect plate washing can lead to inaccurate results. Before adding test solution B or substrate, make sure to absorb the liquid in the well as much as possible. In order to prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test. The enzyme label plate is covered with a cover or film to avoid liquid evaporation; the next step should be performed as soon as possible after washing the plate to avoid the length of the enzyme plate Time is dry.

4. Eliminate the residual liquid and fingerprints on the bottom of the board, otherwise it will affect the OD value.

5. Avoid direct light exposure during storage and incubation.

6. Store unused microplates or reagents at 2-8 ° C. Standard products, test solution A working fluid, and test solution B working fluid should be configured and used according to the required amount. Please accurately configure the standard product and working fluid, and try not to configure it in a small amount (such as when drawing test solution A, it should not be less than 10ul at a time) To avoid concentration errors due to inaccurate dilution; the detection solutions A, B, and substrate solutions should be incubated at 37 ° C for 30 minutes before use; do not reuse the diluted standard and detection solution A Working fluid or detection solution B working fluid.

7. It is recommended to set a double-hole test when testing samples to ensure the accuracy of the test results.

Plate washing method Manual plate washing method: absorb (not to touch the wall) or shake off the liquid in the enzyme plate; place a few layers of water on the experimental table

Paper, with the microtiter plate down and vigorously pat several times; inject at least 0.3ml of the recommended wash buffer into the well and soak for 1-2 minutes, as needed

Yes, repeat this process several times.
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 bone degradation specific marker (CTX-2) at the same time, and has no cross reaction with other related proteins.

Calculation

Take the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as the ordinate (ordinary coordinate), draw a standard curve on semi-logarithmic coordinate paper (recommended to use professional curve making software for analysis, such as curve expert 1.3) , According to the OD value of the sample, find the corresponding concentration from the standard curve, and then multiply it by the dilution factor; or use the standard concentration and OD value to calculate the regression equation of the standard curve, and substitute the OD value of the sample into the equation to calculate the concentration , Multiplied by the dilution factor, which is the actual concentration of the sample.

Precautions

1. The washing process is very important. Insufficient washing can easily cause false positives.

2. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a multi-channel pipette

3. Please make a standard curve at the same time of each measurement, and it is best to make a double hole.

4. 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.

5. When preparing standard products and testing solution working fluids, please prepare with corresponding diluent, not to be confused.

6. Please keep the substrate away from light.

Detection range: 78 pg / ml-5,000 pg / ml

Minimum detection limit: 19.5 pg / 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. 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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