2.6 ELISA Method for Measuring Serum IL-12 Concentration

2.6 ELISA Method for Measuring Serum IL-12 Concentration

Remove the eyeballs of the mice to collect blood, use clean test tubes to collect the blood, let it coagulate at room temperature for 2 hours, centrifuge at 1500 rpm for 10 minutes, collect the serum and package it, then freeze at -20℃ for storage.

IL-12 concentration measurement: Follow the instructions in the kit.

Reagent preparation:

• Standard solution preparation: Dissolve 200μl of distilled water in each tube before use.
• Washing solution: Dilute with twice the amount of distilled water.
• Substrate working solution preparation: Before coloring, dissolve OPD tablets in substrate dilution solution for 5–10 minutes, adding 5ml of solution to each tablet.

Testing procedure:

1. Take the kit out of the refrigerator 20 minutes before the experiment to bring it to room temperature (20–25℃).
2. Take out the required strips from the sealed bags that have been brought to room temperature.
3. Establish a standard curve: Set up 8 standard wells, add 100μl of sample dilution to each well, add 100μl of standard solution to the first well, mix well, then use a pipette to draw 100μl and transfer it to the second well. Continue like this...

Research on Pei Zhengxue's series of formulas

Repeatedly perform serial dilutions until the seventh well, finally drawing 100μl from the seventh well and discarding it, so that all wells contain 100μl. The eighth well serves as the blank control group. Fourth, add samples: Add 100μl of the sample to be tested to each well. Fifth, place the reaction plate at 37℃ for 120 minutes. Sixth, wash the plate: Wash the reaction plate thoroughly with washing solution 4–6 times, then blot dry on filter paper. Seventh, add 50μl of primary antibody working solution to each well. Eighth, mix the reaction plate thoroughly and place it at 37℃ for 60 minutes. Ninth, wash the plate again: Wash the reaction plate thoroughly with washing solution 4–6 times, then blot dry on filter paper. Tenth, add 100μl of enzyme-labeled antibody working solution to each well. Eleventh, place the reaction plate at 37℃ for 60 minutes. Twelfth, wash the plate as before. Thirteenth, add 100μl of substrate working solution to each well, place it at 37℃ in the dark for 5–10 minutes. Fourteenth, add 50μl of termination solution to each well and mix well. Fifteenth, measure the OD value at 450nm using an enzyme reader, and find the corresponding concentration on the coordinate graph based on the absorbance value of the sample.

2.7 Statistical Methods

All experimental data are expressed as mean ± standard deviation (x̄±s), and SPSS 17.0 statistical software is used for data analysis. One-way ANOVA is applied for comparing means across multiple groups. A significance level of P<0.05 is adopted to determine statistically significant differences.

3 Experimental Results