Detection of HLA-G by a specific sandwich ELISA using monoclonal antibodies G233 and 56B

Molecular Human Reproduction Vol.8, No.8 pp. 776–784, 2002 Detection of HLA-G by a specific sandwich ELISA using monoclonal antibodies G233 and 56B M.J.C.van Lierop1, F.Wijnands1, Y.W.Loke4, P.M.Emmer2, H.G.M.Lukassen3, D.D.M.Braat3, A.van der Meer2, S.Mosselman1 and I.Joosten2 1Department of Pharmacology, NV Organon, 5342 CC Oss, Departments of 2Bloodtransfusion and Transplantation Immunology and 3Gynaecology and Obstetrics, University Medical Centre Nijmegen, Nijmegen, The Netherlands and 4Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, Cambridge, UK To whom correspondence should be addressed at: NV Organon, Department of Pharmacology, Room RE 3201, P.O.Box 20, 5340 BH Oss, The Netherlands. E-mail: Human leukocyte antigen (HLA)-G, which is mainly expressed at the maternal–fetal interface, may play a role in the immune tolerance of the semi-allogenic fetus by the mother. Functional studies have shown that HLA-G is indeed a potential modulator of different immune responses. Therefore, it is of interest to study the level of expression of soluble HLA-G in several biological fluids derived from women with and without fertility problems. In order to measure soluble HLA-G, a reliable and sensitive HLA-G specific sandwich ELISA is required. Here, we describe such an ELISA in which G233 is used as the coating antibody and 56B as the detecting antibody. In comparison with two other assays, this assay shows highest responses to recombinant HLA-G and native HLA-G in primary trophoblast culture supernatant and high responses to HLA-G in amniotic fluid. No HLA-G in follicular fluid or preimplantation embryo culture supernatant could be detected. Key words: 56B/BFL.1/ELISA/G233/HLA-G Introduction The non-classical major histocompatibility complex (MHC) class I molecule, human leukocyte antigen (HLA)-G, has gained a lot of interest in reproductive immunology because of its predominant expression in trophoblast cells. HLA-G differs from other MHC class I molecules by its low polymorphism, truncated cytoplasmic tail and the existence of seven splice variants (Geraghty et al., 1987; Ishitani and Geraghty, 1992; Fujii et al., 1994; Kirszenbaum et al., 1994; Paul et al., 2000a). In particular, the full length HLA-G isoform (HLA-G1) has clearly been shown to be expressed at the cell surface or expressed and secreted in a soluble form (HLA-G5) (Bainbridge et al., 2000a; Mallet et al., 2000; Paul et al., 2000a). Cell surface expression of the other membrane-bound isoforms (HLA-G2, -G3 and -G4) has recently also been shown on transfected cells (Riteau et al., 2001). However, whether these shorter isoforms of HLA-G are really expressed under physiological conditions is still a subject of debate (Bainbridge et al., 2000a; Mallet et al., 2000). The restricted expression of HLA-G at the maternal–fetal interface suggests that it may play a major role in the immune tolerance of the semi-allogenic fetus by the mother. Functional studies have shown that HLA-G is a potential modulator of different immune responses. It has the capacity to function as a restriction element for mouse CD8⫹ cytotoxic T cells (Horuzsko et al., 1997). HLA-G has also been shown to form a ligand for several killer inhibitory receptors present on natural killer cells, myelomonocytic cells and/or T cell subsets (Cantoni et al., 1998; Navarro et al., 1999; Pazmany et al., 1999). Through these receptors HLA-G might be able to modulate cytotoxicity, cytokine production and proliferation, as has been shown 776 in vitro (Maejima et al., 1997; Riteau et al., 1999; Bainbridge et al., 2000b; Kapasi et al., 2000). Furthermore, in-vitro studies with soluble HLA-G have shown induction of apoptosis of blast-like cells (Fournel et al., 2000a), an effect on the release of cytokines from peripheral blood mononuclear cells (Kanai et al., 2001) and inhibition of proliferative allo-responses (Lila et al., 2001). If HLA-G is indeed essential for successful embryo implantation and early pregnancy, it is of interest to study the level of expression of soluble HLA-G in several biological fluids derived from women with and without fertility problems. In order to measure soluble HLA-G, different groups have reported the use of ELISA techniques using different monoclonal antibodies. However, for most of these antibodies cross-reactivity with other HLA molecules can not completely be excluded. Alternatively, when no HLA-G specific antibodies were available, indirect methods, including immunodepletions, have been used (Athanassakis et al., 1999; Fournel et al., 1999; Puppo et al., 1999; Rebmann et al., 1999). A study has been described in which different sandwich ELISA methods to detect soluble HLA-G were compared (Fournel et al., 2000b). However, the well characterized, highly HLA-G specific antibody G233 (Loke et al., 1997) was not used in this study. Here, we describe a reliable and sensitive HLA-G specific sandwich ELISA in which G233 is used as the coating antibody and 56B as the detecting antibody. In comparison with two other assays, this assay shows highest responses to recombinant HLA-G and native HLA-G in primary trophoblast culture supernatant and high responses to HLA-G in amniotic fluid. No HLA-G in follicular fluid or preimplantation embryo culture supernatant could be detected. A different assay, in which BFL.1 is used as the detecting © European Society of Human Reproduction and Embryology HLA-G specific sandwich ELISA Figure 1. Western blot of 1% CHAP-lysates from several cell lines stained by 56B. Cells (5⫻107/ml) were lysed in 25 mmol/l Iodoacetamide, 5 mmol/ l sodium orthovanadate, 1% CHAPS and 200 µg/ml PMSF and 5 µl of each lysate was loaded onto the gel. Lane 1: LCL 721.221 lysate, lane 2: LCL 721.221-G (transfected with full length HLA-G), lane 3: low range marker, lane 4: K562 lysate, lane 5: K562-G (transfected with full length HLA-G), lane 6: JEG-3 lysate. After running, the gel was blotted on Hybond ECL nitrocellulose membrane (Amersham). The membrane was blocked by 5% blocking reagent (Amersham) and incubated overnight at 4°C with 2 µg/ml 56B in 0.5% PBST and 10% goat serum. Final steps were performed with second antibody and conjugate from the Elite Vectastain ABC kit (Vector Laboratories) and with ECL solutions and ECL hyperfilm from Amersham. Arrows indicate bands of ~40 kDa in the HLA-G transfected cell lines (lanes 2 and 5) and two bands of ~37 and 39 kDa in the JEG-3 lysate (lane 6). Figure 2. Immunohistochemical staining of a paraffin section of a first trimester human implantation site by 56B. Paraffin sections were subjected to high temperature antigen unmasking in 0.01 mol/l citrate buffer before staining. Antibody incubations were performed at room temperature in a humid atmosphere for 30 min each and sections were washed twice for 5 min in PBS between steps. Sections were preblocked for 20 min with 2% normal horse serum. An optimal dilution of 56B w (...truncated)