Comparative reactivity of different HLA-G monoclonal antibodies to solubleHLA-G molecules

Different HLA-G monoclonal antibodies (mAbs) were first evaluated for their capability to identify soluble HLA-G (sHLA-G) in ELISA. Three of them, nam ely 87G, BFL.1 and MEM-G/9, when used as coating mAbs together with W6/32 c apture mAb, identified beta S-microglobulin (beta 2m)-associated-sHLA-G but not soluble HLA-B7 (sHLA-B7) in cell culture supernatants from transfected cells. By comparison, the anti-HLA class I mAb 90 did recognize both sHLA- G and sHLA-B7. By using these HLA-G mAbS, sHLA-G was identified in amniotic fluids as well as in culture supernatants of first trimester and term plac ental explants but not in cord blood. Intron 4-retaining sHLA-G isoforms we re identified in some amniotic fluids by the use of an intron 4-specific mA b (16G1). Reactivity of these different HLA-G mAbs was then compared to det ermine their respective binding sites on soluble and membrane-bound HLA-G. Using both ELISA and how cytometry analysis, we showed that they did not co mpete with each other which suggested that they did not recognize the same determinants. Finally, we report that two mAbs directed against the al doma in of HLA class I heavy chain (mAb 90 and YTH 862) did compete with 87G, th erefore demonstrating that this latter mAb recognized an epitope localized on this external domain of HLA-G.