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.