| Product Description | The reagent kit adopts a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). Add the sample, standard substance, and HRP labeled detection antibody to the pre coated micropores of adenosine diphosphate (ADP) antibody, then incubate and thoroughly wash. Using substrate TMB for color development, TMB is converted to blue under the catalysis of peroxidase, and finally to yellow under the action of acid. The depth of color is positively correlated with adenosine diphosphate (ADP) in the sample. Measure the absorbance (OD value) using an ELISA reader at a wavelength of 450nm and calculate the sample concentration.
Sample collection, processing, and preservation methods
1. Serum: Use a test tube that does not contain pyrogens and endotoxins. Avoid any cell stimulation during the operation. After collecting blood, centrifuge at 3000 rpm for 10 minutes to quickly and carefully separate the serum and red blood cells.
2. Plasma: EDTA, citrate or heparin anticoagulation. Centrifuge at 3000 rpm for 30 minutes to obtain the supernatant.
3. Cell supernatant: Centrifuge at 3000 rpm for 10 minutes to remove particles and polymers.
4. Tissue homogenization: Add an appropriate amount of physiological saline to the tissue and crush it. Centrifuge at 3000 rpm for 10 minutes to obtain the supernatant.
5. Storage: If the sample is not tested in a timely manner after collection, please divide it into batches according to the amount used, freeze it at -20 ℃, avoid repeated freeze-thaw, thaw it at room temperature, and ensure that the sample is evenly charged and thawed.
Self provided items
1. Enzyme reader (450nm)
2. High precision sampler and nozzle: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
3.37 ℃ constant temperature box
Operation precautions
1. Store the reagent kit at 2-8 ℃ and equilibrate at room temperature for 20 minutes before use. The concentrated washing solution taken out of the refrigerator will have crystals, which is a normal phenomenon. The water bath is heated to completely dissolve the crystals before use.
2. The Flat noodles not used in the experiment shall be immediately put back into the self sealing bag and sealed (low-temperature dry) for storage.
3. S0 standard substance with a concentration of 0 can be considered as a negative control or blank; When operating according to the instructions, the sample has been diluted 5 times, and multiplying the final result by 5 is the actual concentration of the sample.
4. Strictly follow the time, amount of liquid added, and sequence specified in the instruction manual for temperature cultivation.
5. Shake all liquid components thoroughly before use.
Kit composition | 名称 | 96孔配置 | 48孔配置 | 备注 | | 微孔酶标板 | 12孔×8条 | 12孔×4条 | 无 | | 标准品 | 0.3mL*6管 | 0.3mL*6管 | 无 | | 样本稀释液 | 6mL | 3mL | 无 | | 检测抗体-HRP | 10mL | 5mL | 无 | | 20×洗涤缓冲液 | 25mL | 15mL | 按说明书进行稀释 | | 底物A | 6mL | 3mL | 无 | | 底物B | 6mL | 3mL | 无 | | 终止液 | 6mL | 3mL | 无 | | 封板膜 | 2张 | 2张 | 无 | | 说明书 | 1份 | 1份 | 无 | | 自封袋 | 1个 | 1个 | 无 |
注：标准品（S0-S5）浓度依次为：0、400、800、1600、3200、6400 nmol/L
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Preparation of reagents
twenty × Dilution of washing buffer: Dilute distilled water at a ratio of 1:20, which is 20 parts of 1 part × Add 19 parts of distilled water to the washing buffer.
Washing method
1. Manual washing of the board: Shake off the liquid in the holes, fill each hole with washing solution, let it stand for 1 minute, shake off the liquid in the holes, pat dry on absorbent paper, and wash the board 5 times in this way.
2. Automatic plate washer: Inject 350 cleaning solution into each hole μ L. Soak for 1 minute and wash the board 5 times.
Operation steps
1. Take out the required Flat noodles from the aluminum foil bag after 20 min of room temperature balance, and seal the remaining Flat noodles with a self sealing bag and put it back at 4 ℃.
2. Set standard and sample wells, with different concentrations of standard added to each standard well μ L;
3. Add the sample to be tested to the sample well first μ L. Add another sample diluent of 40 μ L; Blank holes are not added.
4. Except for blank wells, add 100 detection antibodies labeled with horseradish peroxidase (HRP) to each well of the standard and sample wells μ L. Seal the reaction pores with a sealing film and incubate in a 37 ℃ water bath or constant temperature box for 60 minutes.
5. Discard the liquid, pat dry on absorbent paper, fill each hole with washing solution, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this process 5 times (the board can also be washed with a washing machine).
6. Add 50 substrates A and B each to each well μ L. Incubate at 37 ℃ in dark for 15 minutes.
7. Add 50 terminating liquids to each well μ L. Measure the OD values of each well at a wavelength of 450nm within 15 minutes.
Result judgment
Draw standard curve: In the Excel worksheet, use the standard concentration as the x-axis and the corresponding OD value as the y-axis to draw the standard linear regression curve. Calculate the concentration values of each sample according to the curve equation. 
Reagent kit performance
1. Accuracy: The correlation coefficient R between the standard linear regression and the expected concentration is greater than or equal to 0.9900.
2. Sensitivity: The minimum detection concentration is less than 10 nmol/L.
3. specificity: does not cross react with other soluble structural analogues.
4. Repeatability: The coefficient of variation within and between boards is less than 15%.
5. Storage: Store at 2-8 ℃, away from light and moisture.
6. Validity period: 6 months
Disclaimers
1. The reagent kit is for research purposes only and shall not be used in clinical or human experiments. Otherwise, all consequences arising from it shall be borne by the experimenter, and our company shall not be responsible.
2. Strictly follow the instructions for operation. If the experimenter violates the instructions, the consequences shall be borne by the experimenter. |
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