Sample Processing and Requirements: Serum: Place whole blood specimens collected in serum separator tubes at room temperature for 2 hours or at 4°C overnight, then centrifuge at 1000×g for 20 minutes. Remove the supernatant, or store at -20°C or -80°C, but avoid repeated freezing and thawing. Plasma: Collect specimens using EDTA or heparin as an anticoagulant and centrifuge at 1000×g for 15 minutes at 2-8°C within 30 minutes of collection. Remove the supernatant for testing, or store at -20°C or -80°C, but avoid repeated freezing and thawing. Tissue Homogenate: Rinse 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 and mince the tissue. Add the minced tissue to a corresponding volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample corresponds to 9mL 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 thoroughly grind on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly freeze-thawed. Finally, centrifuge the homogenate at 5000×g for 5–10 minutes, and collect the supernatant for analysis. Other biological fluids: Centrifuge at 1000xg for 20 minutes, remove the supernatant, and test. Pre-test preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare the standard gradient working solution: Add 1 mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, and then gently mix (concentration is 100 ng/mL). Then dilute to the following concentrations: 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.125 ng/mL, 1.5625 ng/mL, and 0 ng/mL. Serial dilution method: Take 7 EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 100 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 50 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves directly as a blank well; there is no need to aspirate the liquid from the penultimate tube.
- Prepare the biotinylated detection antibody working solution: Centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration with universal diluent (e.g., 10 μL concentrate + 990 μL universal diluent). Prepare freshly for use.
- Prepare the enzyme conjugate working solution: Centrifuge the 100× concentrated enzyme conjugate at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated HRP enzyme conjugate to a 1× working concentration with universal diluent (e.g., 10 μL concentrate + 990 μL universal diluent). Prepare freshly for use. 5. Prepare 1× Wash Buffer: Dissolve 10 mL of 20× Wash Buffer in 190 mL of distilled water (Concentrated Wash Buffer removed from the refrigerator may crystallize, which is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing the 1× Wash Buffer). Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return them to 4°C. 2. Add Samples: Add 100 μL of sample or standard of varying concentrations to the corresponding wells. Add 100 μL of Universal Diluent to the blank wells. Cover with a film sealer and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample to be tested at least 1-fold with Universal Diluent before adding it to the ELISA plate. This reduces the impact of matrix effects on the test results. When calculating the sample concentration, multiply by the corresponding dilution factor. It is recommended to run replicates for all test samples and standards.) 3. Add Biotinylated Antibody: Remove the ELISA plate, discard the liquid, and do not wash. Add 100 μL of biotinylated antibody working solution directly to each well, cover with a film, and incubate at 37°C for 60 minutes. 4. Wash: Discard the liquid and add 300 μL of 1x wash buffer to each well. Let stand for 1 minute, shake off the wash buffer, and pat dry on absorbent paper. Repeat this process three times (a plate washer can also be used). 5. Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well. Cover with a film, and incubate at 37°C for 30 minutes. 6. Wash: Discard the liquid and wash the plate five times according to the washing method in step 4. 7. Add substrate: Add 90 μL of substrate (TMB) to each well, cover with a film, and incubate at 37°C in the dark for 15 minutes. 8. Add stop solution: Remove the ELISA plate and add 50 μL of stop solution directly to each well. Immediately measure the OD value of each well at 450 nm. Calculation of Experimental Results: Interpretation of Results: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as the correction value. Plot the standard curve of the four-parameter logistic function on double-logarithmic graph paper, with concentration as the horizontal axis and OD value as the vertical axis. 2. If the sample OD value is higher than the upper limit of the standard curve, dilute the sample appropriately and retest. Multiply the sample concentration by the corresponding dilution factor.
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