title: An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry.
abstract: HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope. Graphical Abstract ᅟ.
authors.affiliation: Laboratory of Analytical Chemistry, University of Konstanz, D-78457, Konstanz, Germany.|Steinbeis Center for Biopolymer Analysis and Biomedical Mass Spectrometry, D-65428, Rüsselsheim am Main, Germany.
authors.forename: Marius-Ionuţ
authors.initials: MI
authors.lastname: Iuraşcu
authors.affiliation: Division of Oncology, University Hospital Zürich, Rämistr. 100, 8091, Zürich, Switzerland.
authors.forename: Osiris
authors.initials: O
authors.lastname: Marroquin Belaunzanar
authors.affiliation: Laboratory of Analytical Chemistry, University of Konstanz, D-78457, Konstanz, Germany.|Steinbeis Center for Biopolymer Analysis and Biomedical Mass Spectrometry, D-65428, Rüsselsheim am Main, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Cozma
authors.affiliation: Division of Oncology, University Hospital Zürich, Rämistr. 100, 8091, Zürich, Switzerland.
authors.forename: Ulf
authors.initials: U
authors.lastname: Petrausch
authors.affiliation: Department of Biomedicine, University of Basel, Petersgraben 4, 4031, Basel, Switzerland. christoph.renner@hirslanden.ch.
authors.forename: Christoph
authors.initials: C
authors.lastname: Renner
authors.affiliation: Laboratory of Analytical Chemistry, University of Konstanz, D-78457, Konstanz, Germany. michael.przybylski@stw.de.|Steinbeis Center for Biopolymer Analysis and Biomedical Mass Spectrometry, D-65428, Rüsselsheim am Main, Germany. michael.przybylski@stw.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Przybylski
keywords: Affinity mass spectrometry; Affinity quantification; Ankylosing Spondylitis; HLA B27; Mixed-disulfide linked epitope; Proteolytic epitope excision