Recombinant MD2 Protein Binding Assay

The recombinant MD2 (rMD2, 1 μg/mL) (R&D Systems, Minneapolis, MN, USA) protein diluted in PBS was immobilized in 96-well plates (Thermo Fisher Scientific Inc., Waltham, MA, USA) and incubated at 37 °C for 2 h. The wells were washed with PBS, followed by blocking with 300 μL of 5% BSA in PBS at room temperature overnight. The plate was then washed with PBST (PBS with 0.05% Tween-20) [10 (link)]. MMPP and Dex were pre-incubated at 37 °C for 1 h. Then biotinylated LPS (100 ng/mL) (Invivogen, San Diego, CA, USA) was added and incubated at room temperature for 1 h. Streptavidin conjugated to horseradish peroxidase (HRP) was incubated at 37 °C for 1.5 h [37 (link)]. The activity of HRP was determined using KLP SureBlue^TM TMB substrate, and the reaction was stopped by 2.5 N H₂SO₄. The optical density of each well was measured at a wavelength of 450 nm, using a microplate reader (Apollo LB 9110; Berthold Technologies GmbH, Bad Wildbad, Germany).