Soluble HLA-G: purification from eukaryotic transfected cells and detection by a specific ELISA.
OBJECTIVE
The detection of soluble forms of human leukocyte antigen-G molecule (sHLA-G) at the maternal-fetal interface suggest that sHLA-G may play a role during pregnancy. To study the potential functions of sHLA-G, we developed a procedure to detect and produce such HLA-G isoforms.


METHODS
Transfected cell lines expressing either sHLA-G1s cDNA (JAR-G1s) or an sHLA-G monochain DNA (Fox-G-mono) containing extracellular domains of HLA-G linked to the human beta 2-microglobulin were used. Specific sHLA-G enzyme-linked immunosorbent assay (ELISA), using anti-HLA-G monoclonal antibodies (mAbs) (87G and BFL.1) as coating antibodies and the biotinylated HLA class I mAb, W6/32, to reveal the bound molecules, was then developed.


RESULTS
To assess the specificity of the ELISA, we tested cell culture supernatants from the trophoblast-derived JEG-3 cell line and the HLA-G1s-transfected JAR cells, and we detected sHLA-G in both supernatants. sHLA-G monochain was also detected by ELISA in transfected cell supernatants using the conformational mAb, W6/32, showing that the conformation of sHLA-G monochain was proper. Using the same ELISA, sHLA-G was detected in various samples of amniotic fluid. To test the potential role of sHLA-G, sHLA-G has been purified by immunoaffinity columns, using W6/32 mAb, from culture supernatants of HLA-G1s or sHLA-G monochain-transfected cells.


CONCLUSIONS
These important tools will be useful both for the detection of sHLA-G in various biological fluids and in functional tests.
Fournel
S
S
INSERM U395, Toulouse, France.
Aguerre-Girr
M
M
Campan
A
A
Salauze
L
L
Berrebi
A
A
Lone
Y C
YC
Lenfant
F
F
Le Bouteiller
P
P
D000653:Amniotic Fluid / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006680:HLA Antigens / Q000032:analysis* / Q000302:isolation & purification*; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I / Q000032:analysis* / Q000302:isolation & purification*; D006801:Humans; D011233:Precipitin Tests; D011247:Pregnancy; D012680:Sensitivity and Specificity; D012995:Solubility; D014162:Transfection; D014327:Trophoblasts / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D006680:HLA Antigens; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I