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

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Abstract
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.