Soluble HLA-G: Purification from Eukaryotic Transfected Cells and Detection by a Specific ELISA
Article first published online: 6 SEP 2011
American Journal of Reproductive Immunology
Volume 42, Issue 1, pages 22–29, July 1999
How to Cite
Fournel, S., Aguerre-Girr, M., Campan, A., Salauze, L., Berrebi, A., Lone, Y.-C., Lenfant, F. and Bouteiller, P. L. (1999), Soluble HLA-G: Purification from Eukaryotic Transfected Cells and Detection by a Specific ELISA. American Journal of Reproductive Immunology, 42: 22–29. doi: 10.1111/j.1600-0897.1999.tb00461.x
- Issue published online: 6 SEP 2011
- Article first published online: 6 SEP 2011
- Accepted January 6, 1999
- MHC class Ib;
- soluble HLA-G;
- immunoaffinity purification
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.