| 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 solution: Gently wash adherent cells with pre-cooled PBS, then digest with trypsin, and collect the cells after centrifugation at 1000×g for 5 minutes; suspended cells can be directly collected by centrifugation. Wash the collected cells 3 times with pre-cooled PBS, add 150-200uL PBS for every 1×10^6 cells to resuspend (it is recommended to add protease inhibitors to PBS; if the content is very low, the PBS volume can be appropriately reduced) and break the cells by repeated freezing and thawing or ultrasound. Centrifuge the extract at 2-8℃, 1500×g for 10 minutes, and take the supernatant for detection.
Cell culture supernatant: Centrifuge at 1000×g for 20 minutes, take the supernatant for detection, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing.Other biological fluids: Centrifuge at 1000xg for 20 minutes and take the supernatant for testing. Preparation before testing: 1.Please take the reagent kit out of the refrigerator 10 minutes in advance and allow it to equilibrate 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 biotinylated antibody working solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration using universal diluent (e.g., 10uL concentrated solution + 990uL universal diluent). Use on the same day. 4.Preparation of enzyme conjugate working solution: 15 minutes before use, centrifuge the 100x concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100x concentrated HRP enzyme conjugate to a 1x working concentration with universal diluent (e.g., 10uL concentrate + 990uL universal diluent). Use on the same day. 5.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 of the refrigerator may have crystals, which is normal. It can be left at room temperature and shaken evenly. Wait until the crystals are completely dissolved before reconstitution).
Operation steps:
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.Sample Addition: Add 100µl of sample or positive and negative controls to the corresponding wells. Add 100µl of universal diluent to the blank wells. Cover with a film sealer and incubate at 37°C for 1 hour. (Recommendation: Dilute the test sample at least 1-fold with universal diluent before adding it to the ELISA plate to minimize matrix effects. When calculating sample concentration, multiply by the dilution factor. It is recommended to run replicates for all test samples and positive and negative controls.)
3.Add biotinylated antibody: Remove the ELISA plate and discard the liquid without washing. Add 100uL of biotinylated antibody working solution directly to each well, cover with sealing film and incubate at 37℃ for 1 hour.
4.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 process 3 times (you can also use a plate washer to wash the plate).
5.Add enzyme conjugate working solution: Add 100uL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37℃ for 30 minutes.
6.Wash the plate: discard the liquid and follow the washing method in step 4, washing the plate 5 times.
7.Add substrate: Add 90uL of substrate (TMB) to each well, cover with sealing film, and incubate at 37℃ in the dark for 15 minutes.
8.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.
Experimental
Result judgment:
1. The conditions for the test results to be valid are: The average of the positive control wellsODValue＞0.5, average OD of negative control wellsValue <0.2.
2. Test samplesS/PValue ≥ 0.2When the sample is positive, it is judged as positive; the sample is testedS/PValue < 0.2When , it is judged as a negative sample. |
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