Comparative reactivity of different HLA-G monoclonal antibodies to soluble HLA-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, namely 87G, BFL.1 and MEM-G/9, when used as coating mAbs together with W6/32 capture mAb, identified beta2-microglobulin (beta2m)-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 placental explants but not in cord blood. Intron 4-retaining sHLA-G isoforms were identified in some amniotic fluids by the use of an intron 4-specific mAb (16G1). Reactivity of these different HLA-G mAbs was then compared to determine their respective binding sites on soluble and membrane-bound HLA-G. Using both ELISA and flow cytometry analysis, we showed that they did not compete with each other, which suggested that they did not recognize the same determinants. Finally, we report that two mAbs directed against the alpha1 domain of HLA class I heavy chain (mAb 90 and YTH 862) did compete with 87G, therefore demonstrating that this latter mAb recognized an epitope localized on this external domain of HLA-G.
Fournel
S
S
Institut National de la Santé et de la Recherche Médicale U395, Hôpital de Purpan, Toulouse, France.
Huc
X
X
Aguerre-Girr
M
M
Solier
C
C
Legros
M
M
Praud-Brethenou
C
C
Moussa
M
M
Chaouat
G
G
Berrebi
A
A
Bensussan
A
A
Lenfant
F
F
Le Bouteiller
P
P
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D002461:Cell Line, Transformed; D017077:Culture Media, Conditioned / Q000737:chemistry; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D005260:Female; D006680:HLA Antigens / Q000276:immunology* / Q000378:metabolism*; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D011247:Pregnancy; D017434:Protein Structure, Tertiary; D012995:Solubility; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D000939:Epitopes; D006680:HLA Antigens; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I