Detection of anti-β1-AR autoantibodies in heart failure by a cell-based competition ELISA.
BACKGROUND
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.


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.
Corimmun GmbH, Fraunhofer Str 17, 82152 Martinsried, Germany.
Hans-Peter
HP
Holthoff
Stefan
S
Zeibig
Valerie
V
Jahns-Boivin
Johannes
J
Bauer
Martin J
MJ
Lohse
Stefan
S
Kääb
Sebastian
S
Clauss
Roland
R
Jahns
Angela
A
Schlipp
Götz
G
Münch
Martin
M
Ungerer
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D001483:Base Sequence; D002311:Cardiomyopathy, Dilated / Q000097:blood / Q000175:diagnosis / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D061987:Sf9 Cells; D014162:Transfection
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D018342:Receptors, Adrenergic, beta-1