Human zonulin elisa kit

For the non-invasive evaluation of IP, the serum levels of sCD163 were determined using a kit (Cusabio Technology, Houston, TX, USA) [22 (link)], while sMR was measured using an enzyme-linked immunosorbent assay (ELISA) kit (LifeSpan BioSciences, Seattle, WA, USA) [23 (link)]. Zonulin was measured using a human zonulin ELISA kit (Elabscience, Wuhan, China) [24 (link)]. All assays were conducted according to the manufacturer’s instructions. The serum samples were collected at baseline and stored at −80 °C until analyses.

Serum was split into 500 μL aliquots to measure BDNF (R&D Systems, Minneapolis, MN, USA), zonulin (Human Zonulin ELISA Kit, Elabscience, Bethesda, MD, USA), lipopolysaccharide (LPS, LSBio, Seattle, WA, USA), Fatty Acid Binding Protein 2 (FABP2, Human FABP2 Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA), and claudin-5 (Human Claudin-5 ELISA Kit, Novus Biologicals, Littleton, CO, USA).

Serum samples were isolated from peripheral blood collected in gel tubes with clot activator. After collection, samples were incubated for 50 min, centrifuged at 1372 g for 5 min, and stored until zonulin determination. A human Zonulin ELISA Kit (Elabscience, MD, USA) was used to quantify zonulin concentrations. Plates were pre-coated with antibodies to human zonulin, and serum samples and standards were incubated for 1 h, 37 °C. Then, incubation with biotinylated detection antibodies and avidin–horseradish peroxidase conjugate was performed for 30 min. Three washing steps followed to remove unbound and free molecules. The substrate solution was added to each well and incubated for 15 min. The enzyme–substrate reaction was blocked by a stop solution, and the color turned yellow. The optical density was measured in a spectrophotometer at 450 nm. A standard curve was constructed, and zonulin concentrations were calculated by converting the obtained optical density in ng/mL.