Characterization of binding and quantification of human autoantibodies to PDGFRα using a biosensor-based approach

Abstract

Systemic sclerosis (SSc) is a chronic autoimmune disease of the connective tissue. The variety and clinical relevance of autoantibodies in SSc patients have been extensively studied, eventually identifying agonistic autoantibodies targeting the platelet-derived growth factor receptor alpha (PDGFRα), and representing potential biomarkers for SSc. We used a resonant mirror biosensor to characterize the binding between surface-blocked PDGFRα and PDGFRα-specific recombinant human monoclonal autoantibodies (mAbs) produced by SSc B cells, and detect/quantify serum autoimmune IgG with binding characteristics similar to the mAbs. Kinetic data showed a conformation-specific, high-affinity interaction between PDGFRα and mAbs, with equilibrium dissociation constants in the low-to-high nanomolar range. When applied to total serum IgG, the assay discriminated between SSc patients and healthy controls, and allowed the rapid quantification of autoimmune IgG in the sera of SSc patients, with anti-PDGFRα IgG falling in the range 3.20-4.67 neq/L of SSc autoantibodies. The test was validated by comparison to direct and competitive anti-PDGFRα antibody ELISA. This biosensor assay showed higher sensibility with respect to ELISA, and other major advantages such as the specificity, rapidity, and reusability of the capturing surface, thus representing a feasible approach for the detection and quantification of high affinity, likely agonistic, SSc-specific anti-PDGFRα autoantibodies.

Systemic Sclerosis: From Pathophysiology to Novel Therapeutic Approaches

“…For example, agonistic antibodies stimulating the Platelet Derived Growing Factor Receptor α (PDGFRα) are capable of inducing the persistent activation of the intracellular signaling cascade that is usually transiently triggered by PDGF, leading to chronic myofibroblast activation and subsequent ECM accumulation [ 16 , 17 , 18 ]. Unlike the natural, non-stimulatory, anti-PDGFRα autoantibodies, agonistic anti-PDGFRα autoantibodies recognize specific conformational epitopes, largely overlapping with the PDGF binding site, suggesting their pathogenic role in the SSc-specific, unbalanced autoimmune response against cellular antigens [ 19 , 20 , 21 ].…”

Trans-spliced peptides spanning PDGFRα and Adeno-Associated Virus type 5 trigger autoimmune responses in systemic sclerosis patients

“…Sensing surfaces containing recombinant human PDGFRα fused to a poly-histidine tag (rhPDGFRα-His) were prepared as previously described (67). Brie y, upon activation of carboxylate groups with an equimolar mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), rhPDGFRα-His was covalently anchored via the N-terminus of the histidine tail.…”

The Challenges of Developing Biosensors for Clinical Assessment: A Review

“…Generally, the biomarkers are categorized as molecular biomarkers, cellular biomarkers, or imaging biomarkers. The molecular biomarkers commonly used in biosensors are proteins (such as IgG, IgM, antibodies, and proteins) and nucleic acid-based markers (such as RNA, DNA, or KRAS genes) [109][110][111][112][113][114]. The biomarker identification methods based on protein biomarkers and nucleic acids are called proteomics and transcriptomics, respectively [115].…”