2.5 ELISA Method for Determining IL-2 Concentration

2.5 ELISA Method for Determining IL-2 Concentration

Induction of IL-2: The above-prepared splenic lymphocyte suspension was added to a 48-well plate, 0.9 mL per well, along with 100 μL of 1640 culture medium containing ConA (final ConA concentration: 5 μg/mL). The plates were then incubated at 37°C with 5% CO₂ for 36 hours [28], and the supernatant was collected (which is IL-2).

Measurement of IL-2 Concentration: The procedure was carried out according to the kit instructions [29].

Reagent Preparation:

1. Take the reagent kit out of the refrigerator 20 minutes in advance to allow it to equilibrate to room temperature.
2. Dilute the concentrated washing solution with double-distilled water (1:20).
3. Standard Solutions: Add 1.0 mL of standard diluent to the lyophilized standard, let it dissolve completely, then let it stand for 15 minutes to mix thoroughly (2000 pg/mL). Further dilute according to the required concentrations on the standard curve (standard curve concentrations: 7.8, 15.625, 31.25, 62.5, 125, 250 pg/mL).
4. Biotinylated Antibody Working Solution: Dilute the concentrated biotinylated antibody with biotinylated antibody diluent (1:100).
5. Enzyme Conjugate Working Solution: Dilute the concentrated enzyme conjugate with enzyme conjugate diluent (1:50).

Research on Pei Zhengxue’s Series of Formulas

1.0 Operating Steps

① Take out the required strips from the sealed bag that has been brought to room temperature.

② Except for the blank wells, add the samples or different concentrations of standard solutions (100 μL/well) to the corresponding wells, seal the reaction wells with sealing tape, and incubate at 37°C for 90 minutes.

③ Manual washing of the plates: Shake off all the liquid in the wells, add 350 μL of washing solution to each well, let it sit for 30 seconds, then shake off the liquid again and pat dry on thick absorbent paper. Wash the plates five times.

④ Except for the blank wells, add the biotinylated antibody working solution (100 μL/well). Seal the reaction wells with sealing tape, and incubate at 37°C for 60 minutes.

⑤ Wash the plates five times.

⑥ Except for the blank wells, add the enzyme conjugate working solution (100 μL/well). Seal the reaction wells with sealing tape, and incubate at 37°C for 30 minutes.

⑦ Wash the plates five times.

⑧ Add 100 μL of developing reagent to each well, avoid light, and incubate at 37°C for 15–20 minutes. Add 100 μL of stop solution to each well, mix well, and measure the OD450 value immediately (within 5 minutes).

2.6 RT-PCR Method for Determining the Expression Levels of IL-2 and IFN-γ mRNA in Splenic Lymphocytes

Handling of RT-PCR experimental equipment: Soak the required centrifuge tubes, PCR reaction tubes, pipette tips, etc., in a 0.1% DEPC solution for 24 hours, then air-dry them in a clean bench for later use.