Sandwich enzyme linked immunosorbent assay kit

Immediately after collection, AH samples were transferred into sterile plastic tubes (safe-lock microcentrifuge tubes, 1.5 mL) and immediately frozen and stored at −80°C until analysis.
The level of KRT8 in the AH was quantitatively assessed using a sandwich enzyme-linked immunosorbent assay kit (Cloud-Clone Corporation, Houston, TX, USA). The assays were performed according to the manufacturers' protocols. Samples were added into the wells of 96-well microplates, and the plates were then incubated for 2.5 hours at room temperature (RT), followed by gentle shaking for two hours at 37°C. Biotinylated antibodies were incubated for one hour at RT with gentle shaking at 37°C. Horseradish peroxidase‐streptavidin solution was incubated for 45 minutes at RT, followed by gentle shaking for 30 minutes at 37°C. Tetramethylbenzidine dihydrochloride substrates were added to each well for 30 minutes in the dark. The enzyme-substrate reaction was terminated by adding sulphur acid solution, and the color change was measured at a wavelength of 450 nm. The level of KRT8 in the samples was then determined by comparing the optical density of the samples to values on the standard curve.