S. Fournel X. Hue M. Aguerre-Girr C. Solier M. Legros C. Praud-Brethenou M. Moussa G. Chaouat A. Berrebi A. Bensussan F. Lenfant P. Le Bouteiller Comparative reactivity of different HLA-G monoclonai antibodies to soiubie HLA-G moiecuies Key words: Soluble HLA-G: HU-G isoforms; HLA-G monoclonal antibodies; ELiSA Acknowledgments: This work was supported by Institut National de ia Sante et de la Recherche Medicaie, by Association pour ia Recherche sur le Cancer (9489), by Universite Paui Sabatier and by Etablissement Frangais des Greffes. We wish to thank Dr. F. C. Grunnet for providing us with the SHLA-B7 and mHLA-B7 transfectants, Dr. D. Geraghty, Dr. H. Waldmann and Pr. J.R Revillard for their gifts of mAbs, Pr. E. Weiss. Dr. S. Maier. Dr. H, Grosse-Wilde and Dr. V. Rebmann for providing us a caiibrated, standard sHLAG. A. Blascliitz is acknowledged for her helpfui advice in the estabiisment of ELiSA procedure and G. Cassar for the flow cytometry analysis. Received 37 October 1999, revised, accepted for puOllcation 20 Match 2O00 ' Munkseaard 2000 TTssue Antigens . ISSN 0001-2815 nssue Antigens 2000; S6; 510-518 Printed in Denmark . All rights reserved MQstract: Different HLA-G monoclonal antibodies (inAbs) were first evalu- ated for their capability to identify soluble HLA-G (sHLA-G) in ELISA. Three of them, namely 87G, BFL.1 and MEM-G/9, when used as coating mAbs together with W6/32 capture mAb, identified p2-mia'oglobulin (|32m)-associ- ated-sHLA-G but not soluble HLA-B7 (sHLA-B7) in cell culture super- natants from transfected cells. By comparison, the anti-HLA class 1 mAb 90 did recognize both sHLA-G and sHLA-B7. By using these HLA-G mAbs, sHLA-G was identified in amniotic fluids as well as in aiiture supernatants of first trimester and term placental explants but not in cord blond. Intron 4-retaining sHLA-G isoforms were identified in some amniotic fluids by the use of an intnin 4-spedfic mAb {16G1). Reactivity of these different HLA- G mAbs was then compared to determine their respective binding sites on soiubie and membrane-bound HLA-G. Using both ELISA and flow cytome- try analysis, we showed that they did not compete with each other, which suggested that they did not recoipiize the same determinants. Finally, we report that two mAbs directed against the al domain of HLA class I heavy chain (mAb 90 and YTH 862) did compete with 87G. therefore demonstrat- ing that this latter mAb recognized an epitope localized on this external domain of HLA-G. HLA-G class Ib gene is characterized by a low polymorphism, unique structural featui-es and a limited tissue distribution (1, 2). Major structural characteristics of HLA-G are as follows. First, the promoter region of HLA-G differs in many ways from the other major histocompatibitity compiex (MHC) class I genes (3,4). Second, the cytoplasmic domain of HLA-G is shorter than in class Ia mol- ecules (5). Third, the primary HLA-G transcripts are differentially spliced, giving rise to six different isoforms (6-9). Four of them encode membrane-bound products lacking either one or two exter- nal domains, two others generate soluble proteins. These soluble forms of HLA-G (sHLA-G). a full-length sHLA-Gl and a shorter sHLA-G2 form lacking the a2 domain, are encoded by alternatively spliced transcripts that retain intron 4 which contains a stop codon, Authors' S. X. Hue', M. C. Soller'. M. Legros'. C. Praud-Brethenou', M. Mousao'. G. CfiaouBl'. A. BerrebP. A. Bensussan', F. Lenfant'. R Le Boutatl>9r> 'Insmj! Nsilonai de la Sante et lie la Recderche M^icale U395, Hdpital de Purpan, Toulouse. France. 'Biologle de la Relation Matemo Foetale. IMSERM U 131, HOpltai Antoine BeclSre. Cfamart, Frarice. ^Service de Gyrtecclogle Obst^rique. HOpltal U Gisue. Toulouse. Fiance. *Facun6 de M^Oecine de Cr#tell, INSERM U4it8, CrSteil, France Corraapondance to; pn 11 tope Le Bnutelller Insitiul National de la Sant^ et de la Rechetctie Centm HospKalo. Univefsiiaire Oe Purpan, BP 3026 31024 Toulouse Cedax 03 France Fa»: +33 562 748 386 Tel: +33 562 748374 510