title: Studies of a murine monoclonal antibody directed against DARC: reappraisal of its specificity.
abstract: Duffy Antigen Receptor for Chemokines (DARC) plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1), but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR) and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland; INSERM, UMR_S1134, F-75015 Paris, France; Institut National de la Transfusion Sanguine, F-75015 Paris, France; Division of Transplantation Immunology and Mucosal Biology, MRC Centre for Transplantation, King's College London, Guy's Hospital, London, United Kingdom.
authors.forename: Dorota
authors.initials: D
authors.lastname: Smolarek
authors.affiliation: INSERM, UMR_S1134, F-75015 Paris, France; Institut National de la Transfusion Sanguine, F-75015 Paris, France; Universite Paris-Diderot, Sorbonne Paris Cité, F-15013, Paris, France; Laboratory of Excellence GR-Ex, Paris, France.
authors.forename: Claude
authors.initials: C
authors.lastname: Hattab
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland; Institute of Biochemistry and Biophysics, Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Buczkowska
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland.
authors.forename: Radoslaw
authors.initials: R
authors.lastname: Kaczmarek
authors.affiliation: Laboratory of Medical Microbiology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Jarząb
authors.affiliation: INSERM, UMR_S1134, F-75015 Paris, France; Institut National de la Transfusion Sanguine, F-75015 Paris, France; Universite Paris-Diderot, Sorbonne Paris Cité, F-15013, Paris, France; Laboratory of Excellence GR-Ex, Paris, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Cochet
authors.affiliation: INSERM, UMR_S1134, F-75015 Paris, France; Institut National de la Transfusion Sanguine, F-75015 Paris, France; Universite Paris-Diderot, Sorbonne Paris Cité, F-15013, Paris, France; Laboratory of Excellence GR-Ex, Paris, France.
authors.forename: Alexandre G
authors.initials: AG
authors.lastname: de Brevern
authors.affiliation: Laboratory of Microbial Immunochemistry and Vaccines Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Lukasiewicz
authors.affiliation: Laboratory of Microbial Immunochemistry and Vaccines Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Jachymek
authors.affiliation: Laboratory of Microbial Immunochemistry and Vaccines Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Niedziela
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Grodecka
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland; Institute of Physiotherapy, Faculty of Physical Education and Physiotherapy, Opole University of Technology, Opole, Poland.
authors.forename: Kazimiera
authors.initials: K
authors.lastname: Wasniowska
authors.affiliation: INSERM, UMR_S1134, F-75015 Paris, France; Institut National de la Transfusion Sanguine, F-75015 Paris, France; Universite Paris-Diderot, Sorbonne Paris Cité, F-15013, Paris, France; Laboratory of Excellence GR-Ex, Paris, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Colin Aronovicz
authors.affiliation: INSERM, UMR_S1134, F-75015 Paris, France; Institut National de la Transfusion Sanguine, F-75015 Paris, France; Universite Paris-Diderot, Sorbonne Paris Cité, F-15013, Paris, France; Laboratory of Excellence GR-Ex, Paris, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Bertrand
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland; Institute of Physiotherapy, Faculty of Physical Education and Physiotherapy, Opole University of Technology, Opole, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Czerwinski
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D004375:Duffy Blood-Group System / Q000276:immunology*; D000939:Epitopes / Q000276:immunology*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D051379:Mice; D011956:Receptors, Cell Surface / Q000276:immunology*
chemical_list: C473745:ACKR1 protein, human; D000911:Antibodies, Monoclonal; D004375:Duffy Blood-Group System; D000939:Epitopes; D011956:Receptors, Cell Surface