title: Identification of circulating nonclassic human leukocyte antigen G (HLA-G)-like molecules in exudates.
abstract: BACKGROUND
  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 methods using highly specific antibodies. However, results of published studies are in conflict regarding the clinical utility and even the nature of HLA-G present in circulation.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
authors.affiliation: Department of Biochemistry, University Clinic of Navarra, 31008 Pamplona, Spain. agonzaleh@unav.es
authors.forename: Alvaro
authors.initials: A
authors.lastname: Gonzalez
authors.forename: Estibaliz
authors.initials: E
authors.lastname: Alegre
authors.forename: Ainhoa
authors.initials: A
authors.lastname: Arroyo
authors.forename: Jöel
authors.initials: J
authors.lastname: LeMaoult
authors.forename: José Ignacio
authors.initials: JI
authors.lastname: Echeveste
mesh_terms: D000918:Antibody Specificity; D014408:Biomarkers, Tumor / Q000378:metabolism*; D015153:Blotting, Western; D016022:Case-Control Studies; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005122:Exudates and Transudates / Q000378:metabolism*; D005260:Female; D006680:HLA Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D009369:Neoplasms / Q000378:metabolism*; D014162:Transfection; D001613:beta 2-Microglobulin / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D006680:HLA Antigens; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I; D001613:beta 2-Microglobulin