Enzyme-linked immunosorbent assay (ELISA)
The contents of platelet-derived growth factor (PDGF)-BB, interferon
(IFN)-γ and TGF-β1 (R&D Systems Inc., Minneapolis, USA) in the protein
extracts were measured by ELISA according to the manufacturer’s
instructions. Briefly, the protein extracts were lysed with 100
mM Tris (pH: 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100,
and 0.5% sodium deoxycholate. Then, 50 µL of standard, control, or
sample was added to each well of the microplate, which was precoated
with capture antibody; A second HRP-labeled antibody was added and bound
to the antigen-antibody complex.Unbound detection antibody was washed
away. Tetramethylbenzidine (TMB) substrate solution was added to the
wells, and a blue color developed in proportion to the amount of analyte
present in the sample. Color development was stopped, turning the color
in the wells yellow. The absorbance of the color at 450 nm was measured.
The contents of PDGF-BB, IFN-γ and TGFβ1 were calculated
by means of standard curves.