Elisa plates 96 well

Serum samples were obtained from normal control and SSHTN mice. TNF-α concentration was measured using ELISA MAX Standard Set Mouse TNF-α (Biolegend, United States) according to the manufacturer’s protocols. 96-well ELISA plates (Biolegend) were precoated with monoclonal hamster antibody in carbonate buffer and incubated overnight at 4°C. To block non-specific binding and reduce background, the plates were incubated for 1 h at room temperature with the addition of 1% bovine serum albumin (BSA; Sigma-Aldrich) in PBS. Diluted mouse TNF-α standard and serum samples were added to the wells and incubated for 2 h at room temperature. Biotinylated goat polyclonal anti-mouse TNF-α detection antibody was added to the wells with 1 h incubation at room temperature. The bound IgG was detected by incubation with HRP-conjugated avidin, followed by colorimetric detection with TMB substrate solution (Biolegend). The reaction was stopped with 2 N H₂SO₄ after 30 min, and the absorbance was measured at 450 nm using a microplate reader (Remote Sunrise, Tecan, Japan). Absorbance value at 595 nm was used as a reference.

96-well ELISA plates (Biolegend, CA, USA) were coated with 50 µL/well of a 1:100 dilution of anti-human CD81 antibodies (R&D Systems, MN, USA) and incubated overnight at 4 °C. After washing three times with PBS, the plates were blocked with 5% BSA in PBS with 0.05% Tween-20 (PBST) at room temperature for 2 h (50 µL/well). Then, plasma exosome samples (100 µL/well) were added to the plate and incubated overnight at 4 °C. After three washes with PBST, 50 µL of anti-human EphA2 antibodies (0.2 μg/mL, Novus, MN, USA) was added and incubated at 37 °C for 1 h. The plates were then washed three times with PBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (BIORAD, CA, USA) at room temperature for 1 h (100 μL/well). After three times final washes with PBST, plates were incubated with 50 μL/well TMB reagent (Cell Signaling Technology, MA, USA) at room temperature for 10–15 min, followed by the addition of 50 μL/well of stop solution (2 M H₂SO₄). The absorbance was read at 450 nm using a micro-ELISA reader.