Background: The presence of conformational, stimulatory auto-antibodies (ab) against the PDGF receptor (PDGFR) in patients affected by scleroderma (SSc) has been recently questioned. IgG purification procedures and technical problems in cell-based biological assays and in solid phase binding assays may be the sources of artefacts. To solve these issues and to validate the presence of anti-PDGFR antibodies in SSc patients, a simple, specific and reproducible assay is required. Objectives: To develop a solid phase binding assay to detect agonistic anti-PDGFR antibodies in serum samples in order to discriminate between SSc and other conditions. Methods: We engineered plasmids containing full or truncated DNA sequences encoding human alpha or beta PDGFR subunits, tagged with Histidine or FLAG. These DNA constructs were expressed in PDGFR-/- mammalian cell lines and tested for reactivity with human recombinant (r-) PDGF and with murine anti-PDGFR antibodies directed to conformational epitopes of the receptor. Upon assessing their reactivity with these specific ligands, truncated receptors comprising the extracellular PDGFR region and FLAG or His tag were stably expressed in HeLa cells, purified from cell lysates by chromatography with nickel or anti-FLAG resins and analyzed by SDS PAGE and immunoblotting. r-PDGFR were used to capture anti-PDGFR auto-ab in the serum of SSc patients by ELISA or Surface Plasmon Resonance biosensors. Results: A specific r-receptor version was selectively bound by IgG from serum of SSc patients, but not by control IgG. This same r-receptor was used to develop a specific ELISA to discriminate between SSc sera and sera of other patients and controls. The other r-receptors displayed binding with IgG and sera from controls or patients affected by other diseases. Conclusion: The conformation of the recombinant PDGFR (used in the solid phase assays) is crucial for the detection of SSc-specific anti-PDGFR auto-antibodies. We have generated a r-receptor molecule that binds antibodies in SSc sera and discriminates between SSc and other conditions. The solid phase binding assay based on this r-PDGFR might be used as a novel tool for diagnosis of SSc and classification of the clinical subsets of disease and related conditions such as Raynaud's Phenomenon.
Development of a solid phase binding assay for the detection of conformational anti-PDGFR autoantibodies in the serum of patients affected by scleroderma
NACCI, GIULIA;FUNARO, Ada;
2010-01-01
Abstract
Background: The presence of conformational, stimulatory auto-antibodies (ab) against the PDGF receptor (PDGFR) in patients affected by scleroderma (SSc) has been recently questioned. IgG purification procedures and technical problems in cell-based biological assays and in solid phase binding assays may be the sources of artefacts. To solve these issues and to validate the presence of anti-PDGFR antibodies in SSc patients, a simple, specific and reproducible assay is required. Objectives: To develop a solid phase binding assay to detect agonistic anti-PDGFR antibodies in serum samples in order to discriminate between SSc and other conditions. Methods: We engineered plasmids containing full or truncated DNA sequences encoding human alpha or beta PDGFR subunits, tagged with Histidine or FLAG. These DNA constructs were expressed in PDGFR-/- mammalian cell lines and tested for reactivity with human recombinant (r-) PDGF and with murine anti-PDGFR antibodies directed to conformational epitopes of the receptor. Upon assessing their reactivity with these specific ligands, truncated receptors comprising the extracellular PDGFR region and FLAG or His tag were stably expressed in HeLa cells, purified from cell lysates by chromatography with nickel or anti-FLAG resins and analyzed by SDS PAGE and immunoblotting. r-PDGFR were used to capture anti-PDGFR auto-ab in the serum of SSc patients by ELISA or Surface Plasmon Resonance biosensors. Results: A specific r-receptor version was selectively bound by IgG from serum of SSc patients, but not by control IgG. This same r-receptor was used to develop a specific ELISA to discriminate between SSc sera and sera of other patients and controls. The other r-receptors displayed binding with IgG and sera from controls or patients affected by other diseases. Conclusion: The conformation of the recombinant PDGFR (used in the solid phase assays) is crucial for the detection of SSc-specific anti-PDGFR auto-antibodies. We have generated a r-receptor molecule that binds antibodies in SSc sera and discriminates between SSc and other conditions. The solid phase binding assay based on this r-PDGFR might be used as a novel tool for diagnosis of SSc and classification of the clinical subsets of disease and related conditions such as Raynaud's Phenomenon.
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
