2.5 ELISA Method for Detecting Serum IL-12 Concentration

2.5 ELISA Method for Detecting Serum IL-12 Concentration

After enucleating the mice, blood is collected in clean test tubes and allowed to clot at room temperature for 2 hours. Then, the blood is centrifuged at 1500 rpm for 10 minutes to collect the serum, which is stored at -20°C.

1. Prepare standard solutions: Set up 8 standard wells and perform serial dilutions. First, add 100 μl of sample diluent to each well, then add 100 μl of standard solution to the first well, mix well, and use a pipette to draw out 100 μl and transfer it to the second well, mix again, then draw out another 100 μl and transfer it to the third well, and continue this process until the seventh well. Finally, draw out 100 μl from the seventh well and discard it. Each well holds 100 μl, and the eighth well is the blank control group, to establish a standard curve.
2. Add 100 μl of the serum to be tested to each well, then place the plates in a 37°C incubator for 90 minutes.
3. Wash each well 3–5 times thoroughly, then pat dry on filter paper.
4. Prepare the primary antibody working solution, 50 μl per well, and seal the plates tightly with sealing tape, then incubate at 37°C for 60 minutes.
5. Wash each well 3–5 times thoroughly, then pat dry on filter paper.
6. Add 100 μl of the enzyme-labeled antibody working solution to each well, then place the plates in a 37°C incubator for 60 minutes.
7. Wash the plates as before.
8. Add 100 μl of the substrate working solution to each well, then place the plates in a 37°C incubator for 5–10 minutes.
9. Finally, add 50 μl of stop solution to terminate the reaction.
10. Place the reaction plates in the enzyme reader (450 nm) to measure the OD value, and find the concentration on the coordinate graph based on the OD value.