Enzyme-linked immunosorbent assay (ELISA)
ELISA was used to measure the concentration of the target protein.
Briefly, 96-well plates were coated with capture antibody and left
overnight at 4°C. Plates were then washed with PBST and blocked with 3%
BSA solution for 1 hour at room temperature. Plates were washed again
and samples, standards and controls were added to the wells. After 2
hours of incubation at room temperature, the plates were washed again
and detection antibodies were added. After incubation with the detection
antibody for 1 h at room temperature, the plate was washed and then
treated with substrate solution (Wuhan jiyinmei Biotechnology Co., Ltd)
for 20 min. Finally, the stop solution was added and the absorbance at
450 nm was measured using a microplate reader (Redu). The concentration
of the target protein in the sample was determined by comparing the
absorbance of the sample to a standard curve. All experiments were
performed in triplicate.