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

Abstract

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 pathologyassociated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinitymass 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, and , as well as the homo-and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants K D towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a K D of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

Increased level of H-ferritin and its imbalance with L-ferritin, in bone marrow and liver of patients with adult onset Still's disease, developing macrophage activation syndrome, correlate with the severity of the disease

“…HC10 recognizes b2m-free heavy chain forms of most HLA-B and some HLA-C alleles [35]. HD6 has a greater specificity for B27 homodimers [5,39]. Both HC10-and HD6-reactive forms of NC-B27 have been shown to bind to the killer immunoglobulin-like receptor KIR3DL2 [20,23].…”

Molecular Epitope Determination of Aptamer Complexes of the Multidomain Protein C‐Met by Proteolytic Affinity‐Mass Spectrometry

“…The PROTEX-SPR-MS method has been successfully applied in a number of recent studies for epitope identifications of protein-antibody complexes, protein-peptide interactions, and carbohydrate-protein complexes. [26][27][28][29][30][31][32] SPR determinations of the C-Met-aptamer complexes revealed high affinities, consistent with the suitability of aptamers as specific inhibitors of C-Met. PROTEX-MS using immobilized aptamer affinitymatrices revealed specific epitopes on C-Met, with a linear epitope peptide identified for the CLN0004 aptamer, and a discontinuous epitope comprising two specific peptides for the CLN0003 aptamer.…”

“…For the identification of aptamer epitopes of C‐Met and determination of binding affinities, selective proteolytic extraction of C‐Met‐aptamer complexes in combination with electrospray (ESI) and MALDI mass spectrometry, and SPR (surface plasmon resonance) biosensor analysis were used as principal tools (PROTEX‐SPR‐MS) (Supplementary Figure S1). The PROTEX‐SPR‐MS method has been successfully applied in a number of recent studies for epitope identifications of protein‐antibody complexes, protein‐peptide interactions, and carbohydrate‐protein complexes . SPR determinations of the C‐Met‐aptamer complexes revealed high affinities, consistent with the suitability of aptamers as specific inhibitors of C‐Met.…”

“…Epitope analysis was carried out by epitope-extraction mass spectrometry, as previously described. [29,30] Briefly, 10 μg of the tryptic digestion mixture was incubated with the CLN0003 aptamer affinity column. An aliquot digest mixture of 10 μg was incubated in the same manner with the CLN0004 aptamer.…”

Antibody Epitope of Human α‐Galactosidase A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease

“…We have recently developed an online surface plasmon resonance (SPR) biosensing technique combined with MS, which enables simultaneous affinity isolation, structure identification and affinity quantification of epitopes from an immobilized antibody–ligand complex, using an integrated, automated interface for sample concentration and in situ desalting for MS analysis . The PROTEX‐MS method, using either proteolytic epitope excision of intact immune complexes or epitope extraction from a defined peptide fragment mixture presented to the immobilized antibody, has been successfully applied to the identification of both linear and discontinuous epitopes of a wide range of protein antigens . In general, the PROTEX‐MS method is based on 1) the high stability of antibodies toward protease digestion, and 2) the proteolytic shielding of the epitope–paratope interaction structure …”