Tmb substrate reagent

Dex-FITC in the sera was measured using a custom-built ELISA. Ninety-six well plates were coated with anti-FITC monoclonal antibody (1F8-1E4, Invitrogen) at 4°C overnight and blocked with 1% bovine serum albumin (BSA), 0.05% Tween 20, PBS. Mouse sera were 1/10 diluted with PBS and added to the plate and incubated at room temperature for 2 h. After washing with PBST, the plate was incubated with a biotin-conjugated anti-FITC monoclonal antibody (NAWESLEE, eBioscience) for 1 h, followed by streptavidin-horseradish peroxidase (R&D systems) for 20 min. TMB substrate reagent (R&D systems) was then added to each well and the plate was incubated for a further 30 min; 2N H₂SO₄ solution was added to stop the reaction. The absorbance at 450 nm was measured using a Multiskan FC multiplate photometer (Thermo Fisher Scientific), and the basal absorbance at 570 nm was subtracted from the values.

Acetonitrile (Sigma), Antibodies: peroxidase (HRP)-conjugated affinity pure goat anti-mouse IgG Fcγ-specific, (Jackson ImmunoResearch); bovine serum albumin BSA (SERVA); Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore Inc.), sodium thiocyanate NaSCN (Sigma); sequencing grade modified porcine trypsin (Promega), TMB substrate reagent (R&D).