BACKGROUND: Autoantibodies against β1-adrenergic receptors (β1AR) that stimulate cardiac cAMP-production play a causal role in the pathogenesis of human heart failure. Patients can be subjected to specific therapies, if the presence of potentially cardio-noxious β1AR-autoantibodies is reliably diagnosed. This requires assessment of IgG-interactions with the native β1AR because β1AR-autoantibodies target a conformational epitope inadequately presented by denatured receptors or linear peptides. Here, we report on a standardised diagnostic procedure for the assessment of β1AR-autoantibodies in heart failure patients, which is based on IgG-binding to native human β1AR. METHODS: Good laboratory practice (GLP)-conform measurement of β1AR-autoantibodies was based on flow-cytometric quantification of differential IgG-binding to native HT1080 cells overexpressing biofluorescent human β1AR or not. Receptor-specific IgG-binding was derived from IgG-related median fluorescence of β1AR-positive cells corrected for background staining of β1AR-negative cells admixed to each measurement. The slope of IgG binding at two different concentrations was used as measure for the titre/avidity of β1AR-autoantibodies. RESULTS: Sensitivity and specificity of the novel procedure for high β1AR-autoantibody levels in dilated cardiomyopathy patients (n=40, NYHA class III-IV) relative to n=40 matched healthy subjects was >90%. It was similar to functional assays considered the gold standard and vastly superior to existing screening-procedures employing fixed cells or linear receptor-peptides as auto-antigenic targets. Inter-assay scatter was 7%-15% and linear dilution recovery was within ±10% of expected values throughout. CONCLUSIONS: The novel assay possibly provides a tool to determine true prevalence and clinical impact of β1AR-autoantibodies. Furthermore, it may serve as companion diagnostic for therapies specifically directed at β1AR-autoantibodies.
BACKGROUND: Autoantibodies against β1-adrenergic receptors (β1AR) that stimulate cardiac cAMP-production play a causal role in the pathogenesis of humanheart failure. Patients can be subjected to specific therapies, if the presence of potentially cardio-noxious β1AR-autoantibodies is reliably diagnosed. This requires assessment of IgG-interactions with the native β1AR because β1AR-autoantibodies target a conformational epitope inadequately presented by denatured receptors or linear peptides. Here, we report on a standardised diagnostic procedure for the assessment of β1AR-autoantibodies in heart failurepatients, which is based on IgG-binding to native human β1AR. METHODS: Good laboratory practice (GLP)-conform measurement of β1AR-autoantibodies was based on flow-cytometric quantification of differential IgG-binding to native HT1080 cells overexpressing biofluorescent human β1AR or not. Receptor-specific IgG-binding was derived from IgG-related median fluorescence of β1AR-positive cells corrected for background staining of β1AR-negative cells admixed to each measurement. The slope of IgG binding at two different concentrations was used as measure for the titre/avidity of β1AR-autoantibodies. RESULTS: Sensitivity and specificity of the novel procedure for high β1AR-autoantibody levels in dilated cardiomyopathypatients (n=40, NYHA class III-IV) relative to n=40 matched healthy subjects was >90%. It was similar to functional assays considered the gold standard and vastly superior to existing screening-procedures employing fixed cells or linear receptor-peptides as auto-antigenic targets. Inter-assay scatter was 7%-15% and linear dilution recovery was within ±10% of expected values throughout. CONCLUSIONS: The novel assay possibly provides a tool to determine true prevalence and clinical impact of β1AR-autoantibodies. Furthermore, it may serve as companion diagnostic for therapies specifically directed at β1AR-autoantibodies.
Authors: Diana Ernst; Johan Westerbergh; Georgios Sogkas; Alexandra Jablonka; Gerrit Ahrenstorf; Reinhold Ernst Schmidt; Harald Heidecke; Lars Wallentin; Gabriela Riemekasten; Torsten Witte Journal: Sci Rep Date: 2019-10-10 Impact factor: 4.379