title: 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.
abstract: 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.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Bornholz
authors.forename: Thomas
authors.initials: T
authors.lastname: Benninghaus
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Reinke
authors.forename: Stephan B
authors.initials: SB
authors.lastname: Felix
authors.forename: Dirk
authors.initials: D
authors.lastname: Roggenbuck
authors.forename: Valérie
authors.initials: V
authors.lastname: Jahns-Boivin
authors.forename: Roland
authors.initials: R
authors.lastname: Jahns
authors.forename: Fritz
authors.initials: F
authors.lastname: Boege
mesh_terms: D000328:Adult; D000368:Aged; D001323:Autoantibodies / Q000097:blood* / Q000276:immunology*; D002311:Cardiomyopathy, Dilated / Q000097:blood / Q000175:diagnosis* / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry / Q000592:standards*; D006801:Humans; D008297:Male; D008875:Middle Aged; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics* / Q000276:immunology*; D012680:Sensitivity and Specificity
chemical_list: D001323:Autoantibodies; D018342:Receptors, Adrenergic, beta-1