Identification of circulating nonclassic human leukocyte antigen G (HLA-G)-like molecules in exudates

HLA-G in biological fluids has been proposed to be useful as a tumor marker as both a diagnostic and prognostic factor. Most HLA-G measurement procedures are based on ELISA

The main HLA-G antigenic molecules in exudates are HLA-G-like complexes, a factor that should be considered when analyzing HLA-G in biological fluids.

We collected 118 exudates, 94 from cancer patients and 24 from patients without tumors. We measured HLA-G concentrations by ELISA using MEM-G/9 or G233 as capture antibody. Samples were immunoprecipitated with an anti-HLA-G antibody and analyzed by Western blot using a different anti-HLA-G antibody.

Discrepancies in HLA-G concentrations in exudates were observed depending on what capture anti-HLA-G antibody was used for ELISA (r = 0.376). These discrepancies were not observed when the ELISAs were performed using culture supernatants from HLA-G1-transfected cells (r = 0.983). Immunoprecipitation and Western blot of cell culture supernatants with 2 different anti-HLA-G antibodies produced the typical band at 39 kDa assigned to HLA-G. When the immunoprecipitation and western blot were performed with exudates, however, there were bands at 53 kDa and 70-76 kDa, higher molecular weights than those usually assigned to HLA-G. These HLA-G-like molecules were associated with β(2)-microglobulin and could also form disulfide bridges with other HLA-G-like molecules. Conclusions: The main HLA-G antigenic molecules in exudates are HLA-G-like complexes, a factor that should be considered when analyzing HLA-G in biological fluids.