A standardised FACS assay based on native, receptor transfected cells for the clinical diagnosis and monitoring of β1-adrenergic receptor autoantibodies in human heart disease

TY - JOUR

T1 - A standardised FACS assay based on native, receptor transfected cells for the clinical diagnosis and monitoring of β1-adrenergic receptor autoantibodies in human heart disease

AU - Bornholz, Beatrice

AU - Benninghaus, Thomas

AU - Reinke, Yvonne

AU - Felix, Stephan B

AU - Roggenbuck, Dirk

AU - Jahns-Boivin, Valérie

AU - Jahns, Roland

AU - Boege, Fritz

PY - 2016/4

Y1 - 2016/4

N2 - 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.

AB - 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.

KW - Adult

KW - Aged

KW - Autoantibodies

KW - Cardiomyopathy, Dilated

KW - Female

KW - Flow Cytometry

KW - Humans

KW - Male

KW - Middle Aged

KW - Receptors, Adrenergic, beta-1

KW - Sensitivity and Specificity

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1515/cclm-2015-0603

DO - 10.1515/cclm-2015-0603

M3 - SCORING: Journal article

C2 - 26408610

VL - 54

SP - 683

EP - 691

JO - CLIN CHEM LAB MED

JF - CLIN CHEM LAB MED

SN - 1434-6621

IS - 4

ER -