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. 2016 Jun;27(6):1105-12.
doi: 10.1007/s13361-016-1361-9. Epub 2016 Apr 11.

An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

Marius-Ionuţ Iuraşcu  1   2 , Osiris Marroquin Belaunzanar  3 , Claudia Cozma  1   2 , Ulf Petrausch  3 , Christoph Renner  4 , Michael Przybylski  5   6
Affiliations

An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

Marius-Ionuţ Iuraşcu et al. J Am Soc Mass Spectrom. 2016 Jun.

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.

Keywords: Affinity mass spectrometry; Affinity quantification; Ankylosing Spondylitis; HLA B27; Mixed-disulfide linked epitope; Proteolytic epitope excision.

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