Detection of Anti–β1-AR Autoantibodies in Heart Failure by a Cell-Based Competition ELISA

Abstract

Rationale: Autoantibodies directed against the second extracellular loop of the cardiac β1-adrenergic receptor (β1-AR) are thought to contribute to the pathogenesis of dilated cardiomyopathy (DCM) and Chagas heart disease. Various approaches have been used to detect such autoantibodies; however, the reported prevalence varies largely, depending on the detection method used. Objective: We analyzed sera from 167 DCM patients (ejection fraction <45%) and from 110 age-matched volunteers who did not report any heart disease themselves, with an often used simple peptide-ELISA approach, and compared it with a novel whole cell–based ELISA, using cells expressing the full transgene for the human β1-AR. Additionally, 35 patients with hypertensive heart disease with preserved ejection fraction were investigated. Methods and Results: The novel assay was designed according to the currently most reliable anti-TSH receptor antibody-ELISA used to diagnose Graves disease (“third-generation assay”) and also detects the target antibodies by competition with a specific monoclonal anti–β1-AR antibody (β1-AR MAb) directed against the functionally relevant β1-AR epitope. Anti–β1-AR antibodies were detected in ≈60% of DCM patients and in ≈8% of healthy volunteers using the same cutoff values. The prevalence of these antibodies was 17% in patients with hypertensive heart disease. Anti–β1-AR antibody titers (defined as inhibition of β1-AR MAb-binding) were no longer detected after depleting sera from IgG antibodies by protein G adsorption. In contrast, a previously used ELISA conducted with a linear 26-meric peptide derived from the second extracellular β1-AR loop yielded a high number of false-positive results precluding any specific identification of DCM patients. Conclusions: We established a simple and efficient screening assay detecting disease-relevant β1-AR autoantibodies in patient sera yielding a high reproducibility also in high throughput screening. The assay was validated according to “good laboratory practice” and can serve as a companion biodiagnostic assay for the development and evaluation of antibody-directed therapies in antibody-positive heart failure.