Research paper A novel sandwich ELISA for a1 domain based detection of soluble HLA-G heavy chains Herbert Juch a , Astrid Blaschitz a , Christine Daxbo ¨ck a , Christine Rueckert b , Kristina Kofler a , Gottfried Dohr a, * a Institute of Cell Biology, Histology and Embryology, Center of Molecular Medicine, Medical University of Graz, Austria, Harrachgasse 21, A-8010 Graz, Austria b Institut fu ¨ r Immungenetik, Charite ´-Universitaetsmedizin Berlin, Campus Virchow-Klinikum, Berlin, Germany Received 7 January 2005; received in revised form 8 September 2005; accepted 22 September 2005 Available online 4 November 2005 Abstract The detection of soluble human leukocyte antigen G (HLA-G) has been a technically demanding task for several years now and various enzyme linked immunosorbent assay (ELISA) formats have been designed. However, no ELISA test has been described so far which is able to detect all possible kinds of soluble HLA-G (sHLA-G) molecules that might occur in bio fluids. Here we describe a new ELISA approach able to recognize soluble a1 domain containing heavy chains of all HLA-G isoforms. The detection limit is shown to be at about 150 pg soluble recombinant HLA-G1 heavy chain per milliliters. Detectable HLA-G fragments are shown to occur in the supernatants of different HLA-G transfected cell lines and appear to be particularly abundant in supernatant of trophoblast derived choriocarcinoma cell lines. The novel ELISA employs the well characterized HLA-G mAbs 4H84 and MEM-G1 which ensure high HLA-G specificity. A negative control ELISA format, designed against non-existing analytes, has been established to reveal non-specific signal interference. D 2005 Elsevier B.V. All rights reserved. Keywords: Soluble HLA-G; ELISA; Heavy chain; 4H84; MEM-G/1; Interference 1. Introduction 1.1. HLA-G The nonclassical HLA class 1b molecule HLA-G has been investigated for nearly 20 years now and has nevertheless remained a mystery. On the one hand it shares several features with the classical HLA class 1a antigens, being a peptide presenting heterodimeric gly- coprotein, non-covalently associated with h 2 -microglo- bulin (h2m). On the other hand HLA-G exhibits some unique properties. It is characterized by a very low polymorphism, a truncated cytoplasmic tail and a re- stricted tissue distribution (for a review see Bainbridge et al., 2001). HLA-G is physiologically expressed at the feto-ma- ternal interface (McMaster et al., 1995), and the main function of HLA-G is thought to be its involvement in the protection of the semiallogenic fetal trophoblast cells against maternal NK-cell mediated immune attack 0022-1759/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2005.09.016 Abbreviations: biot., biotinylated; BSA, bovine serum albumin; h2m, h 2 -microglobulin; ELISA, enzyme-linked immunosorbent assay; HAAA, human anti animal antibodies; Ig, immunoglobulin; HLA, human leukocyte antigen; mAb, monoclonal mouse antibody; OD, optical density; PBS, phosphate buffered saline; RT, room tem- perature; sHLA-G, soluble HLA-G. * Corresponding author. Tel.: +43 316 380 4230; fax: +43 316 380 9625. E-mail address: gottfried.dohr@meduni-graz.at (G. Dohr). Journal of Immunological Methods 307 (2005) 96 – 106 www.elsevier.com/locate/jim