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.