| Usage | Experimental equipment required for the experiment:
1. Microplate reader (450nm)
2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL
3. 37℃ constant temperature box
4. Distilled water or deionized water
Sample processing and requirements:
Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge the specimen at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. Tissue homogenization: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results). Weigh the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL of PBS. The specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000 × g for 5-10 minutes, and the supernatant can be assayed. Cell Lysis Buffer: Gently wash adherent cells with pre-chilled PBS, then trypsinize and collect them by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash the collected cells three times with pre-chilled PBS and resuspend them in 150-200 μL of PBS per 1×10^6 cells (it is recommended to add protease inhibitors to the PBS; if the cell count is very low, the PBS volume can be reduced appropriately). Disrupt the cells by repeated freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and remove the supernatant for analysis. Cell culture supernatant: Centrifuge at 1000×g for 20 minutes, remove the supernatant, or store at -20°C or -80°C, but avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge at 1000×g for 20 minutes, and remove the supernatant for analysis.Please take the test kit out of the refrigerator 10 minutes in advance and equilibrate it to room temperature. 2.Preparation of positive and negative control working solutions: Add 1 mL of universal diluent to each freeze-dried control, let stand for 15 minutes until completely dissolved, and then gently mix. 3.Preparation of HRP-secondary antibody working solution: 15 minutes before use, centrifuge 100× concentrated HRP-secondary antibody at 1000×g for 1 minute, and dilute 100× concentrated HRP-secondary antibody to 1× working concentration with universal diluent (e.g. 10uL concentratedReduction solution + 990uL universal diluent), ready for use. 4.Preparation of 1× washing solution: Take 10mL of 20× washing solution and add it to 190mL of distilled water (the concentrated washing solution taken out from the refrigerator may have crystals, which is normal. It can be placed at room temperature and shaken evenly until the crystals are complete.Prepare after it is completely dissolved).
1.After equilibration at room temperature for 10 minutes, remove the required strips from the aluminum foil bag and seal the remaining strips in a ziplock bag and place them back at 4°C.
2.Add sample: add 100uL of sample or positive and negative control into the corresponding wells respectively.Add 100uL of universal diluent to the white wells. Cover with the sealing film and incubate at 37℃ for 60 minutes. (Recommendation: Dilute the sample to be tested with universal diluent at least 1 times before adding it to the ELISA plate for testing. This will reduce the risk ofTo minimize the impact of matrix effects on test results, the sample concentration should be multiplied by the corresponding dilution factor when calculating the final sample concentration. It is recommended to set up duplicate wells for all test samples and positive and negative controls during the test).
3.Wash the plate: discard the liquid, add 300uL 1x washing solution to each well, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 3 times (you can also use a plate washer to wash the plate).
4.Add HRP-secondary antibody: add 100uL HRP-secondary antibody working solution to each well, cover with sealing film and incubate at 37℃ for 30 minutes.
5.Wash the plate: discard the liquid and follow the washing method in step 3, wash the plate 5 times
6.Add substrate: Add 90uL substrate (TMB) to each well, cover with sealing film, and incubate at 37℃ in the dark for 15 minutes.
7.Add stop solution: Take out the ELISA plate and add 50uL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450nm.
Experiment
Result evaluation:
1.The conditions for the test results to be valid are: The average of the positive control wellsODValue＞0.20, average OD of negative control wellsValue < 0.20.
2.Test samplesS/PValue ≥ 0.2When the sample is positive, it is judged as positive;S/PValue < 0.2, it is judged as a negative sample. |
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