Detection of DCM-associated β1-adrenergic receptor autoantibodies requires functional readouts or native human β1-receptors as targets

Bornholz, Beatrice; Hanzen, Birgit; Reinke, Yvonne; Felix, Stephan B.; Schimke, Ingolf; Wallukat, Gerd; Boege, Fritz

doi:10.1016/j.ijcard.2015.10.068

Cited by 17 publications

(21 citation statements)

References 9 publications

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“…This classical bioassay has recently been transferred to a fully automated high throughput level by Joshi-Barr et al [13] . A FACS assay, based on native beta1-adrenoceptor conformation [14] , also proved to be appropriate and comparable to a high degree to the previously mentioned so-called “classical” bioassay of beta1-AAB measurement which is based on the spontaneously beating neonatal rat cardiomyocytes [15] . Other tests which are based on a biological read-out used receptor-mediated changes of contractility of small arterioles (in vitro) as successfully used and described by Li et al in 2014 [11] .…”

Section: Introductionmentioning

confidence: 74%

Performance and in-house validation of a bioassay for the determination of beta1-autoantibodies found in patients with cardiomyopathy

Wenzel

Schulze-Rothe

Haberland

et al. 2017

Heliyon

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BackgroundAutoantibodies specific for the adrenergic beta1-receptor were identified to be an essential factor for the pathogenesis of dilated cardiomyopathy. For the detection of these autoantibodies, a bioassay was developed and has been used, measuring the positive chronotropic effect on spontaneously beating neonatal rat cardiomyocytes. In order to use this bioassay as an analytical tool to monitor the effectiveness of autoantibody neutralizing therapy in a regulated field, there is a need to assess its analytical performance and validate it according to current guidelines.MethodsUsing standard autoantibody samples, the increased beat rate compared to the basal rate [delta beats/min] was recorded when investigating guideline required assay performance parameters.ResultsThe analytical specificity and sensitivity of the bioassay was demonstrated. The limit of detection and positivity cut-off level were determined to be 3.56 and 7.97 delta beats/min, respectively. The coefficient of variation (CV) of all tested single values (four technical replicates each) was ≤15.2%. The CV of precision within each measuring series did not exceed 20%. Furthermore, the sample stability under a variety of different storage conditions was assessed, as well as the robustness of the cardiomyocyte preparations, which were both given.ConclusionThis bioassay fulfilled guideline determined quality requirements and proved to be appropriate for its application in clinical trials.

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Section: Introductionmentioning

confidence: 74%

Performance and in-house validation of a bioassay for the determination of beta1-autoantibodies found in patients with cardiomyopathy

Wenzel

Schulze-Rothe

Haberland

et al. 2017

Heliyon

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“…This substance was later proven to be a β 1 R-AAb. β 1 R-AAbs have been reported to be a common target for CHF caused by several autoantibody-associated diseases, including DCM, Chagas disease and atrial fibrillation (21)(22)(23). Previous studies by our group indicated that, in patients with CHF arising from different causes, the positive rates of β 1 R-AAbs were all significantly higher compared with those of healthy subjects and were associated with the severity of CHF (24,25).…”

Section: Discussionmentioning

confidence: 90%

Diagnostic and prognostic value of autoantibodies against β1‑adrenoreceptors in patients with heart failure following acute myocardial infarction: A 5‑year prospective study

Wang

Han

et al. 2019

Exp Ther Med

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“…To date, DCM-relevant anti-β 1 -aabs can only be detected by native assays or functional readouts. 19,24,25 However, the many patients potentially to be screened for anti-β 1 -aabs (i) require clinical diagnostics based on conventional procedures [e.g. enzyme-linked immunosorbent assay (ELISA)] for measuring IgG binding to synthetic representations of the very auto-epitope conformation specifically targeted in the course of allosteric β 1 -AR activation.…”

Section: Introductionmentioning

confidence: 99%

“…enzyme-linked immunosorbent assay (ELISA)] for measuring IgG binding to synthetic representations of the very auto-epitope conformation specifically targeted in the course of allosteric β 1 -AR activation. 17,24 Interestingly, circular peptide representations of β 1 -EC II (alone or as an add-on to cardioselective β 1 -blockers) could stop progression and even revert CHF in rats subjected to β 1 EC II immunization(s), whereas cardioselective β 1 -blockers alone were only able to stabilize the dilated cardiomyopathic phenotype. 19,20 Therefore, cyclopeptides might provide an adequate synthetic mimic of the DCM-relevant conformational auto-epitope.…”

Section: Introductionmentioning

confidence: 99%

Unmasking features of the auto‐epitope essential for β₁‐adrenoceptor activation by autoantibodies in chronic heart failure

et al. 2020

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Aims Chronic heart failure (CHF) can be caused by autoantibodies stimulating the heart via binding to first and/or second extracellular loops of cardiac β 1-adrenoceptors. Allosteric receptor activation depends on conformational features of the autoantibody binding site. Elucidating these features will pave the way for the development of specific diagnostics and therapeutics. Our aim was (i) to fine-map the conformational epitope within the second extracellular loop of the human β 1adrenoceptor (β 1 EC II) that is targeted by stimulating β 1-receptor (auto)antibodies and (ii) to generate competitive cyclopeptide inhibitors of allosteric receptor activation, which faithfully conserve the conformational auto-epitope. Methods and results Non-conserved amino acids within the β 1 EC II loop (compared with the amino acids constituting the EC II loop of the β 2-adrenoceptor) were one by one replaced with alanine; potential intra-loop disulfide bridges were probed by cysteine-serine exchanges. Effects on antibody binding and allosteric receptor activation were assessed (i) by (auto)antibody neutralization using cyclopeptides mimicking β 1 EC II ± the above replacements, and (ii) by (auto)antibody stimulation of human β 1-adrenoceptors bearing corresponding point mutations. With the use of stimulating β 1-receptor (auto)antibodies raised in mice, rats, or rabbits and isolated from exemplary dilated cardiomyopathy patients, our series of experiments unmasked two features of the β 1 EC II loop essential for (auto)antibody binding and allosteric receptor activation: (i) the NDPK 211-214 motif and (ii) the intra-loop disulfide bond C 209 ↔C 215. Of note, aberrant intra-loop disulfide bond C 209 ↔C 216 almost fully disrupted the functional auto-epitope in cyclopeptides. Conclusions The conformational auto-epitope targeted by cardio-pathogenic β 1-receptor autoantibodies is faithfully conserved in cyclopeptide homologues of the β 1 EC II loop bearing the NDPK 211-214 motif and the C 209 ↔C 215 bridge while lacking cysteine C 216. Such molecules provide promising tools for novel diagnostic and therapeutic approaches in β 1-autoantibodypositive CHF.

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Detection of DCM-associated β1-adrenergic receptor autoantibodies requires functional readouts or native human β1-receptors as targets

Cited by 17 publications

References 9 publications

Performance and in-house validation of a bioassay for the determination of beta1-autoantibodies found in patients with cardiomyopathy

Performance and in-house validation of a bioassay for the determination of beta1-autoantibodies found in patients with cardiomyopathy

Diagnostic and prognostic value of autoantibodies against β1‑adrenoreceptors in patients with heart failure following acute myocardial infarction: A 5‑year prospective study

Unmasking features of the auto‐epitope essential for β₁‐adrenoceptor activation by autoantibodies in chronic heart failure

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Detection of DCM-associated β1-adrenergic receptor autoantibodies requires functional readouts or native human β1-receptors as targets

Cited by 17 publications

References 9 publications

Performance and in-house validation of a bioassay for the determination of beta1-autoantibodies found in patients with cardiomyopathy

Performance and in-house validation of a bioassay for the determination of beta1-autoantibodies found in patients with cardiomyopathy

Diagnostic and prognostic value of autoantibodies against β1‑adrenoreceptors in patients with heart failure following acute myocardial infarction: A 5‑year prospective study

Unmasking features of the auto‐epitope essential for β1‐adrenoceptor activation by autoantibodies in chronic heart failure

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Unmasking features of the auto‐epitope essential for β₁‐adrenoceptor activation by autoantibodies in chronic heart failure