Soluble HLA-G: Purification from Eukaryotic Transfected Cells and Detection by a Specific ELISA


INSERM U 395, CHU purpan, BP 3028, 31024 Toulouse Cedex 03, France.


PROBLEM: 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.

METHOD OF STUDY: 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 β2-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.

CONCLUSION: These important tools will be useful both for the detection of sHLA-G in various biological fluids and in functional tests.