2.6.2 TNF-α Concentration Determination

2.6.2 TNF-α Concentration Determination

Operate strictly according to the kit instructions.

Reagent Preparation: ① Take the kit out of the refrigerator 30 minutes in advance to allow it to reach room temperature. ② Dilute the standard solution: Add 1 ml of sample diluent to the freeze-dried standard tube, dissolve thoroughly, then perform serial dilutions. ③ Prepare the biotin-labeled antibody working solution: Calculate the total amount needed based on 0.1 ml per well, and mix 10 μl of biotin-labeled antibody with 990 μl of antibody diluent. ④ Prepare the avidin-peroxidase complex (ABC) working solution: Calculate the total amount needed based on 0.1 ml per well, and mix 10 μl of avidin-peroxidase complex (ABC) with 990 μl of ABC diluent.

Operating Steps: ① Take out the required number of plates from the sealed bag that has been brought to room temperature. ② Add 0.1 ml of each serially diluted standard solution to a row of 8 wells, leaving one well filled only with sample diluent as a control, and add 100 μl of the processed sample to each well. ③ Seal the reaction wells with sealing tape, incubate at 37°C for 90 minutes. ④ Wash the plates manually: Shake off all 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, pat dry on thick absorbent paper, and wash the plates twice. ⑤ Add the prepared biological antibody working solution 0.1 ml per well, one after another. Seal the reaction wells with sealing tape, incubate at 37°C for 60 minutes. ⑥ Wash the plates 3 times with 0.01 M TBS, soaking for about 1 minute each time. ⑦ Add the prepared ABC working solution 0.1 ml per well, one after another. Seal the reaction wells with sealing tape, incubate at 37°C for 30 minutes. ⑧ Wash the plates 5 times with 0.01 M TBS, soaking for about 1–2 minutes each time. ⑨ Add 0.09 ml of TMB developing solution per well, which has been balanced at 37°C for 30 minutes, and incubate at 37°C in the dark. During the reaction, observe frequently; when you can clearly see a gradient of blue in the first 3–4 wells of the standard solution, but the difference becomes less obvious in the last 3–4 wells, add 0.1 ml of TMB stop solution per well (the developing reaction should not exceed 30 minutes). Use a microplate reader to measure the OD value at 450 nm, and find the corresponding concentration on the coordinate graph based on the sample’s absorbance value.

2.7 Determination of Serum IFN-γ Concentration in Tumor-Bearing Mice Using ELISA Method