4.2. sEH Protein Level Quantification

The sEH level in PBMCs was measured using an ultrasensitive PolyHRP based immunoassay, as described elsewhere [102]. Briefly, the high-binding microplate (Nunc Cat.No. 442404) was coated with anti-human sEH rabbit serum (1:2000 dilution) in 0.05 M pH 9.6 carbonate-bicarbonate buffer (100 μL/well) overnight at 4 °C. After washing, the plate was blocked with 3% (w/v) skim milk (300 μL/well) in PBS for one hour and washed before sample application. Serial concentrations of human sEH standards or samples with different dilutions in PBS containing 0.1 mg/mL bovine serum albumin (BSA) were then added to the wells (100 μL/well). To minimize the variation of incubation, human sEH standards and samples were first loaded into a 2-mL 96 deep well microplate (Costar 3960), and then transferred to the microplate for immunoassay within 1 min using a multi-channel pipette. Immediately, biotinylated nanobodies selected for reaction with the human sEH (1 μg/mL, 100 μL/well) in PBS were added to each well. The immunoreaction was allowed to proceed for 1 h. After washing, SA-PolyHRP in PBS (25 ng/mL, 100 μL/well) was added and the reaction continued for another 30 min. After the final washing, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (100 μL/well) was added and the plate was incubated for 10–15 min. After stopping the color development with 2 M of sulfuric acid (100 μL/well), the optical density was recorded at 450 nm within 10 min. All incubations unless otherwise specified were performed at room temperature with shaking (600 rpm); each washing step involved three washings with PBS containing 0.05% Tween-20 (PBST, 300 μL/well). Since the reaction involved two separate sEH-targeting antibodies, one of which was a monoclonal nanobody, the assay was exceptionally selective and sensitive.