Estimation of soluble HLA-G blood serum concentrations
sHLA-G1/G5 protein concentration was estimated in the blood sera of pregnant women delivering SGA or AGA infants. Sandwich enzyme immunoassay (ELISA) (Exbio, Praha, Czech Republic) kit was obtained commercially, and sHLA-G was measured as per the manufacturer’s protocol. A β2- microglobulin (β2m)-associated form of sHLA-G1/G5 is detected in Units/ml. The limit of detection as per the recommended kit is 0.6U/ml. The samples were evaluated in duplicates, with calibrators and blank samples on each plate. Blood sera samples were diluted 4 times using the dilution Buffer (60 μl samples to 180μl dilution Buffer) provided with the kit. 100μl of diluted sera samples were placed in duplicates onto the microtiter plate pre-coated with MEM-G/9 (monoclonal antibody, anti-HLA-G1/G5). The plate was incubated overnight at 4°C. 100μl of monoclonal antihuman β2-microglobin antibody labeled with horseradish peroxidase (HRP) was added to each well post washing steps with the washing buffer. The plate was then incubated for one hour at room temperature on shaker (300rpm). 100μl of substrate solution with tetramethylbenzidine (TMB) was added to each well post the washing steps. With a last incubation step at room temperature for 25 min without shaking, 100μl of acidic stop solution was added to each well. The plate was then analyzed at 450 nm with reference wavelength 630 nm on a microplate reader.