2.6 ELISA Method for Measuring Serum EGF Concentration

2.6 ELISA Method for Measuring Serum EGF Concentration

The eyeballs of the mice were removed to collect blood, which was collected in clean test tubes and allowed to clot at room temperature for 2 hours. After centrifugation (1500 rpm) for 10 minutes, the serum was collected and sealed in Eppendorf tubes, then frozen at -20°C for storage.

EGF concentration measurement: Follow the instructions in the kit.

Reagent preparation:

① Take the kit out of the refrigerator 30 minutes in advance to allow it to equilibrate to room temperature;

② Standard solution dilution: Add 1 ml of sample diluent to the lyophilized standard vial, dissolve thoroughly, then perform serial dilutions;

③ Biotin-labeled antibody working solution: Calculate the total volume based on 0.1 ml per well, prepare by mixing 10 μl of biotin-labeled antibody with 990 μl of antibody diluent;

④ Preparation of avidin-peroxidase complex (ABC) working solution: Calculate the total volume based on 0.1 ml per well, prepare by mixing 10 μl of avidin-peroxidase complex (ABC) with 990 μl of ABC diluent.

Operating steps:

① Take the required strips out of the sealed bags that have already equilibrated to room temperature;

② Add 0.1 ml of each serially diluted standard solution sequentially into a row of 7 wells, leaving one well filled only with sample diluent as a control; 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 Manual plate washing: Shake off all liquid in the wells, add 350 μl of washing solution to each well, let stand for 30 seconds, then shake off the liquid again, pat dry on thick absorbent paper, wash the plates twice;

⑤ Add the prepared biological antibody working solution 0.1 ml per well sequentially, seal the reaction wells with sealing tape, and incubate at 37°C for 60 minutes;

⑥ Wash with 0.01 M TBS three times, soaking for about 1 minute each time;

⑦ Add the prepared ABC working solution 0.1 ml per well sequentially, seal the reaction wells with sealing tape, and incubate at 37°C for 60 minutes;

⑧ Wash with 0.01 M TBS five times, soaking for about 2 minutes each time;

⑨ Add the prepared TMB chromogenic solution 0.09 ml per well sequentially, having already equilibrated at 37°C for 30 minutes, and incubate at 37°C in the dark, observing frequently during the reaction. When the first 3–4 wells of the standard solution show obvious blue gradients, while the last 3–4 wells show little difference, add 0.1 ml of TMB stop solution per well (chromogenic

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reaction should not exceed 30 minutes). Measure the OD value at 450 nm using an enzyme reader, and find the corresponding concentration on the coordinate graph based on the sample's absorbance value.

2.7 Radioimmunoassay Method for Measuring Serum IL-1 Concentration

The eyeballs of the mice were removed to collect blood, which was collected in clean test tubes and allowed to clot at room temperature for 2 hours. After centrifugation (1500 r/min) for 10 minutes, the serum was collected and sealed in Doff tubes, then frozen at -20°C for storage. This experiment uses the equilibrium method: take polypropylene test tubes numbered, and follow the steps in the radioimmunoassay kit manual as shown in the table below:

IL-1β RIA Liquid Addition Procedure (Unit: μL)

Mix thoroughly, incubate at 4°C for 16 hours

Immune separation agent 500 500 500 500

Mix thoroughly, let stand at room temperature for 20 minutes, then centrifuge at 3500 rpm for 25 minutes, discard the supernatant, and measure the cpm count on the γ counter, using a pre-edited program to calculate the sample's IL-1β concentration.

2.8 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 multiple groups' means. A p-value less than 0.05 is considered statistically significant.

3 Experimental Results