ELISA
Soluble PD-1L concentrations in the samples (female patient and healthy
donor serum) were measured by an ELISA kit (ThermoFisher, Waltham,
Massachusetts, USA), according to the manufacturer’s protocol. Briefly,
50 µL of sample in duplicate was added to the sample wells. Wells were
then incubated with the Biotin-Conjugated antibody for 2 h at room
temperature. Streptavidin-HRP was next added to all the samples and
incubated 1 h at room temperature. TMB Substrate Solution was added and
the wells were incubated for about 30 min at room temperature. Finally,
the reaction was stopped with Stop Solution and the color intensity was
measured at 450 nm.