Abstract
Background: Nine proteins were identified as putative profibrotic biomarkers in systemic sclerosis (SSc) and an unrelated fibrotic disease in a previously published proteomic study. As the majority of these proteins were orphans of commercially available antibodies, the nine proteins were investigated to determine whether binding peptide aptamers of the Stefin A quadruple mutant-Tracy variant (referred to as “affimers”) could be validated by enzyme linked immunosorbant assay (ELISA) to allow the quantification of these candidate biomarkers in the sera of SSc patients. Materials and Methods: Candidate biomarker peptides were analysed by high throughput affimer microarray to identify binding affimers. Two candidate biomarkers were prioritised, and binding affimers were expressed from genetically modified BL21 competent E. coli strains and purified. These affimers were used in indirect ELISA, and then sandwich ELISA formats against the candidate biomarker recombinant proteins osteonectin and pigment epi-thetlium-derived factor (PEDF). Results: 39 affimers were identified as binders for eight of the nine candidate biomarker peptides were by affimer microarray; six for osteonectin and eleven for PEDF. Two of the six and all eleven were able to recognize physiological concentrations (5 and 1 μg·ml﹣1) of osteonectin and PEDF, respectively by indirect ELISA. In sandwich ELISA format: two affimers were able to detect recombinant PEDF; however, the two affimers identified in indirect ELISA were unable to recognise recombinant osteonectin, and were thus hypothesised to bind to osteonectin at the same binding site. Discussion: SSc is currently an orphan of fully validated biomarkers, which is required for the development of stratified medicine in this field. This approach has laid the groundwork for an affimer based on multiplexed assay, to validate biomarkers in the sera of SSc patients in the future.
Highlights
Fibrotic disorders, which include systemic sclerosis (SSc) and the newly recognized nephrogenic systemic fibrosis (NSF), are conditions resulting from the abnormal production and deposition of collagen and other extracellular matrix proteins by aberrantly activated fibroblasts [14]
NSF is in a non-autoimmune, non-vasculopathic condition resulting from exposure to gadolinium-containing contrast agents used in magnetic resonance imaging (MRI), in patients with reduced renal function [3,6]
All reagents were incubated for 1 h at room temperature, with washing with PBST [v/v] 0.05% four times between incubations. 50 μl 3,3’,5,5’-tertramethl-benzidine (TMB) was added and reaction arrested with 50 μl 0.5 M hydrochloric acid (HCl) after observable colour change and optical density measured at 450 nm (OD450) by Multiskan EX plate reader and Ascent software
Summary
Fibrotic disorders, which include systemic sclerosis (SSc) and the newly recognized nephrogenic systemic fibrosis (NSF), are conditions resulting from the abnormal production and deposition of collagen and other extracellular matrix proteins by aberrantly activated fibroblasts [14]. NSF is in a non-autoimmune, non-vasculopathic condition resulting from exposure to gadolinium-containing contrast agents used in magnetic resonance imaging (MRI), in patients with reduced renal function [3,6]. These conditions are unrelated to many of their clinical features and underlying aetiology, but have a shared alteration to a profibrotic fibroblast phenotype [3]. Two candidate biomarkers were prioritised, and binding affimers were expressed from genetically modified BL21 competent E. coli strains and purified These affimers were used in indirect ELISA, and sandwich ELISA formats against the candidate biomarker recombinant proteins osteonectin and pigment epithetlium-derived factor (PEDF). In sandwich ELISA format: two affimers were able to detect recombinant PEDF; the two affimers identi-
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