Detection of Anti β1-AR Auto-Antibodies in Heart Failure by a Cell-Based Competition ELISA
Rationale: Autoantibodies directed against the second extracellular loop of the cardiac beta1-adrenergic receptor (β1-AR) are thought to contribute to the pathogenesis of dilated cardiomyopathy (DCM) and Chagas' heart disease. Various approaches have been employed to detect such autoantibodies; however, the reported prevalence varies largely depending on the utilized detection method.
Objective: We analysed 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 employed simple peptide-ELISA approach, and compared it to a novel whole cell-based ELISA using cells expressing the full transgene for the human β1-AR. Additionally, 35 patients with hypertensive heart disease (HHD) 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-β1AR antibody (β1-AR MAb) directed against the functionally relevant β1AR epitope. Anti-β1-AR antibodies were detected in ~60% of DCM patients and in ~8 % of healthy volunteers using the same cut-off values. The prevalence of these antibodies was 17% in patients with HHD. 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 ß1-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” (GLP), and can serve as a companion bio-diagnostic assay for the development and evaluation of antibody-directed therapies in antibody-positive heart failure.
- Received May 10, 2012.
- Accepted July 18, 2012.
- Copyright © 2012, American Heart Association