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A novel recombinant antibody specific to full-length stromal derived factor-1 for potential application in biomarker studies.

ABSTRACT: BACKGROUND:Stromal derived factor-1? (SDF-1?/CXCL12) is a chemokine that is up-regulated in diseases characterised by tissue hypoxia, including myocardial infarction, ischaemic cardiomyopathy and remote ischaemic conditioning (RIC), a technique of cyclical, non-injurious ischaemia applied remote from the heart that protects the heat from lethal ischaemia-reperfusion injury. Accordingly, there is considerable interest in SDF-1? as a potential biomarker of such conditions. However, SDF-1? is rapidly degraded and inactivated by dipeptidyl peptidase 4 and other peptidases, and the kinetics of intact SDF-1? remain unknown. METHODS & RESULTS:To facilitate investigation of full-length SDF-1? we established an ELISA using a novel recombinant human antibody we developed called HCI.SDF1. HCI.SDF1 is specific to the N-terminal sequence of all isoforms of SDF-1 and has a comparable KD to commercially available antibodies. Together with a detection antibody specific to the ?-isoform, HCI.SDF1 was used to specifically quantify full-length SDF-1? in blood for the first time. Using RIC applied to the hind limb of Sprague-Dawley rats or the arms of healthy human volunteers, we demonstrate an increase in SDF-1? using a commercially available antibody, as previously reported, but an unexpected decrease in full-length SDF-1? after RIC in both species. CONCLUSIONS:We report for the first time the development of a novel recombinant antibody specific to full-length SDF-1. Applied to RIC, we demonstrate a significant decrease in SDF-1? that is at odds with the literature and suggests a need to investigate the kinetics of full-length SDF-1? in conditions characterised by tissue hypoxia.

SUBMITTER: Bromage DI

PROVIDER: S-EPMC5381782 | biostudies-other | 2017

REPOSITORIES: biostudies-other

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A novel recombinant antibody specific to full-length stromal derived factor-1 for potential application in biomarker studies.

Bromage Daniel I DI Taferner Stasa S Pillai Mahesh M Yellon Derek M DM Davidson Sean M SM

PloS one 20170405 4

<h4>Background</h4>Stromal derived factor-1α (SDF-1α/CXCL12) is a chemokine that is up-regulated in diseases characterised by tissue hypoxia, including myocardial infarction, ischaemic cardiomyopathy and remote ischaemic conditioning (RIC), a technique of cyclical, non-injurious ischaemia applied remote from the heart that protects the heat from lethal ischaemia-reperfusion injury. Accordingly, there is considerable interest in SDF-1α as a potential biomarker of such conditions. However, SDF-1α ...[more]

PMID: 28379992

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Project description:Rheumatoid arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures (ELS) supporting B cell autoreactivity toward locally generated citrullinated (cit) antigens, including those contained in neutrophil extracellular traps (NETs). However, only a minority of RA-rmAbs from B cells isolated from ELS+ RA tissues react against NETs. Thus, alternative cellular sources of other potential autoantigens targeted by locally differentiated B cells remain undefined. RA fibroblast-like synoviocytes (FLS) have been implicated in the release of RA-associated autoantigens. In this study, we aimed to define stromal-derived autoantigens from RA-FLS targeted by RA-rmAbs. Seventy-one RA-rmAbs were screened toward RA-FLS by living-cell immunofluorescence (IF). Western blotting was used to identify potential autoantigens from RA-FLS protein extracts. Putative candidates were validated using colocalization immunofluorescence confocal microscopy, ELISA, immunoprecipitation assay, and surface plasmon resonance on unmodified/cit proteins. Serum immunoreactivity was tested in anti-citrullinated peptide/protein Abs (ACPA)+ versus ACPA- RA patients. Ten out of 71 RA-rmAbs showed clear reactivity toward RA-FLS in immunofluorescence with no binding to NETs. One stromal-reactive RA-rmAb (RA057/11.89.1) decorated a ∼58-kDa band that mass spectrometry and Western blotting with a commercial Ab identified as calreticulin (CRT). Confocal microscopy demonstrated significant cellular colocalization between anti-CRT RA057/11.89.1 in RA-FLS. RA057/11.89.1 was able to immunoprecipitate rCRT. Deimination of CRT to cit-CRT moderately increased RA057/11.89.1 immunoreactivity. cit-CRT displayed increased blocking capacity compared with unmodified CRT in competitive binding assays. Finally, anti-cit-CRT Abs were preferentially detected in ACPA+ versus ACPA- RA sera. We identified a synovial B cell-derived RA-rmAb locally differentiated within the ELS+ RA synovium reacting toward CRT, a putative novel autoantigen recently described in RA patients, suggesting that FLS-derived CRT may contribute to fuel the local autoimmune response.

Project description:Rituximab, a murine-human chimera, is the first monoclonal antibody (mAb) developed as a therapeutic agent to target CD20 protein. Its Fab domain and its interaction with CD20 have been extensively studied and high-resolution atomic models obtained by X-ray diffraction or cryo-electron microscopy are available. However, the structure of the full-length antibody is still missing as the inherent protein flexibility hampers the formation of well-diffracting crystals and the reconstruction of 3D microscope images. The global structure of rituximab from its dilute solution is here elucidated by small-angle X-ray scattering (SAXS). The limited data resolution achievable by this technique has been compensated by intensive computational modelling that led to develop a new and effective procedure to characterize the average mAb conformation as well as that of the single domains. SAXS data indicated that rituximab adopts an asymmetric average conformation in solution, with a radius of gyration and a maximum linear dimension of 52 Å and 197 Å, respectively. The asymmetry is mainly due to an uneven arrangement of the two Fab units with respect to the central stem (the Fc domain) and reflects in a different conformation of the individual units. As a result, the Fab elbow angle, which is a crucial determinant for antigen recognition and binding, was found to be larger (169°) in the more distant Fab unit than that in the less distant one (143°). The whole flexibility of the antibody has been found to strongly depend on the relative inter-domain orientations, with one of the Fab arms playing a major role. The average structure and the amount of flexibility has been studied in the presence of different buffers and additives, and monitored at increasing temperature, up to the complete unfolding of the antibody. Overall, the structural characterization of rituximab can help in designing next-generation anti-CD20 antibodies and finding more efficient routes for rituximab production at industrial level.

Dataset Information

A novel recombinant antibody specific to full-length stromal derived factor-1 for potential application in biomarker studies.

Publications

A novel recombinant antibody specific to full-length stromal derived factor-1 for potential application in biomarker studies.

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