Detection of soluble HLA-G molecules in plasma and amniotic fluid

Although the cDNA sequence of HLA-G antigens is compatible with their expression as soluble molecules (sHLA-G), the determination of native sHLA-G levels in body fluids has not yet been described. The lack of this information is likely to reflect the difficulties in developing an assay suitable to measure sHLA-G antigens in the presence of soluble HLA-A, -B and -C (sHLA-I) antigens, since most of the available anti-HLA-G mAb do not detect soluble beta2-m associated HLA-G antigens or crossreact with sHLA-I antigens. Therefore, we have developed a two-step assay which eliminates the interference of classical HLA class I antigens. In the first step, the sample is depleted of sHLA-I antigens and of HLA-E antigens with mAb TP25.99. Then, HLA-G antigens are captured with mAb W6/32 and detected with anti-beta2-m mAb in ELISA. Utilizing this assay, sHLA-G antigen levels were measured in EDTA plasma from 92 controls with known HLA types, 28 women at delivery and the corresponding cord bloods and in 50 amniotic fluids. Mean sHLA-G plasma levels did not differ between males (24.9+/-3.0 SEM ng/ml; n=42) and females (20.1+/-2.1 SEM ng/ml; n = 50). However, sHLA-G levels in HLA-A11 positive probands (mean: 13.0+/-4.4 SEM ng/ml; n=12) were significantly (P<0.05) lower than in HLA-A11 negative ones (mean: 24.5+/-2.0 SEM ng/ml; n=80). sHLA-G levels in women at delivery (mean: 22.9+/-2.2 SEM ng/ml; n=28) were in the range of controls but were significantly (P<0.001) reduced in the corresponding cord bloods (mean: 13.8+/-1.5 SEM ng/ml; n=28). sHLA-G levels in amniotic fluids (mean: 15.5 + 1.0 SEM ng/ml; n=50) were significantly (P<0.001) lower than in plasma. sHLA-G levels were 5 and 11% of those of sHLA-I antigens in plasmas and amniotic fluids, respectively. Individual sHLA-G levels were not correlated with sHLA-I levels. SDS-PAGE analysis of plasma sHLA-G antigens revealed two molecular variants with a 35 kD and a 27 kD MW corresponding to the sizes of sHLA-G1 and -G2 isoforms. In conclusion, our study has shown that the two-step assay we have developed is reliable in measuring sHLA-G antigen levels. This assay will facilitate the analysis of the biological and clinical significance of sHLA-G antigens in plasma.