Quantification of Biomarkers in Samples

IL-9, EGFR, AREG, and HIF-1α levels were measured by commercial enzyme-linked immunosorbent assay (ELISA) kits purchased from R&D Systems (Minneapolis, MN, USA), including Human IL-9 DuoSet ELISA (Catalog Number: DY209-05), Human EGFR DuoSet ELISA (Catalog Number: DY231), Human Amphiregulin DuoSet ELISA (Catalog Number: DY262), and Human/Mouse Total HIF-1 alpha/HIF1A DuoSet IC ELISA (Catalog Number: DYC1935-2). Briefly, 100 µL of samples or standards were added to the wells of plates and incubated for 2 h at room temperature. The plates were washed five times. One hundred microliters of the detection antibodies were added to each well and incubated for 2 h at room temperature. The plates were washed five times. One hundred microliters of streptavidin-horseradish peroxidases were added to each well and incubated at room temperature for 20 min. The plates were washed five times. One hundred microliters of substrate solutions were added to each well and incubated at room temperature for 20 min. Fifty microliters of stop solutions were added to each well. The optical density of each well was determined using a microplate reader set to 450 nm.