2.3. Soluble HLA‐G determination

For sHLA‐G determination, samples were thawed at room temperature and centrifuged at 14 000 rpm for 4 minutes. The level of soluble HLA‐G1/HLA‐G5 molecules in the plasma samples was determined by a commercially available sandwich enzyme‐linked immunosorbent assay (ELISA) (EXBIO, Praha, Czech Republic) according to the manufacturer's instructions. This ELISA specifically detects soluble HLA‐G1 and HLA‐G5 in a β2‐microglobulin‐associated form. The limit of detection was 0.6 units/mL. The standard curve ranged from 3.9 to 125 units/mL. Samples were tested in the assay at 1:5 and 1:10 dilution, using dilution buffer 1 of the kit. Subsequently, samples were measured at different dilutions to remain in the linear part of the standard curve (ranging from 1:2 to 1:100).

Samples were run in duplicate and mean absorbance was measured at 450 nm wavelength using a BIO‐RAD Microplate Reader and Microplate Manager 6 software (Hercules, California). Calculations were performed according to the manufacturer's guidelines. Standard curves based on the absorbance of calibrators of known concentrations were used for the determination of sHLA‐G concentration in the samples of interest. Results were expressed as units/mL.