Detailed ELISA Assay Protocol for LPS Detection

A 96-well immunoplate was precoated with 10 μg/mL poly-L-lysine (100 μL per well). ELISA was performed as described previously [35 (link)]. Briefly, the 96-well immunoplate was coated for 2 h with diluted LPS at 37 °C and then washed three times with Wash Buffer (PBS + 0.05% Tween 20). The plates were patted dry and Blocking Buffer (PBS + 5% milk powder) was added to each well followed by incubation at 37 °C for 2 h. After drying the plates, diluted serum from the immunized mice was added to each well and the plate was incubated at 37 °C for 1 h. After another washing and drying step, 1:15,000 diluted HRP-conjugated goat anti-mouse IgG antibody (Abcam, Shanghai, China) was added to each well and incubated at 37 °C for 1 h. The washing and drying step was repeated. The Soluble TMB Kit (CWbio, Beijing, China) was used to initiate the detection reaction. Stop solution (2 M H₂SO₄) was added to each well to stop the reaction, and the absorbance of each well was measured at a wavelength of 490 nm with a microplate reader.