Questionable Validity of Peptide-Based ELISA Strategies in the Diagnostics of Cardiopathogenic Autoantibodies That Activate G-Protein-Coupled Receptors

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

“…Moreover, we demonstrate here and have previously shown [1,7] that chemical denaturation disrupts the epitope(s) targeted by DCM-associated β 1 AR-autoantibodies. Therefore, immune-assays based on denatured or synthetic epitope representations have to be considered unreliable [23], and an assay based on native cells seems an unavoidable pre-requisite for valid and meaningful assessments of clinically relevant β 1 AR-autoantibodies in humans. Among various procedures established for that purpose [8,12,13,28,29] the here proposed method has a realistic chance to be implemented as a standard diagnostic test, because it is (i) based on a stable cell line that can be propagated ad infinitum without the sacrifice of animals, (ii) uses an analytical platform usually present in clinical routine laboratories, and (iii) can be GLP-standardised.…”

“…Compared with clinical data, the latter methods appear by far more valid [7,12,22]. Therefore, peptide-based immunoassays are currently not considered as reliable tools for the assessment of clinically relevant β 1 AR-autoantibodies in human patients [1,4,23].…”

Difference between beta1-adrenoceptor autoantibodies of human and animal origin—Limitations detecting beta1-adrenoceptor autoantibodies using peptide based ELISA technology

“…While research has progressed, the development of drugs designed to neutralize the autoantibodies pathogenic function is comparatively slow, which is mostly owed to the limitations of the analytics of such autoantibodies [ 4 ]. Thus far, functional cell-based bioassays show the most reliable results compared to target-solid-phase-based analytics [ 5 , 6 ]. Although applicable in animal experiments [ 7 , 8 ], experiments using human sample material with peptide-based ELISAs are questionable [ 5 ].…”

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

“…The low titre of beta1-adrenoceptor autoantibodies (beta1-AAB) makes an adequate analysis very challenging. So far it has been reported that analytics, based on the exploitation of the autoantibody functionality are superior over tests, using immobilized target moieties especially of peptide origin [ 6 , 7 ]. Despite intensive efforts, the development of tests which could replace the functional bioassay have often failed.…”

“…Despite intensive efforts, the development of tests which could replace the functional bioassay have often failed. A peptide-based ELISA strategy seems to be questionable [7] . With respect to bioassays, several tests have been developed [ 8 , 9 , 10 , 11 , 12 ] of which the “classical bioassay” of spontaneously beating neonatal rat cardiomyocytes, developed by Wallukat and Wollenberger as early as 1987 [9] , is one of the pioneering tests of the detection of a variety of agonistic acting autoantibodies, including beta1-AAB.…”