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 beta 2-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-beta 2-m mAb in ELISA. Utilizing this assay, sHLA-G anti gen 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 +/- 1.1 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). sHaA-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.