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

Katrin Wenzel, Sarah Schulze-Rothe, Annekathrin Haberland, Johannes Müller, Gerd Wallukat, Hanna Davideit, Katrin Wenzel, Sarah Schulze-Rothe, Annekathrin Haberland, Johannes Müller, Gerd Wallukat, Hanna Davideit

Abstract

Background: Autoantibodies 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.

Methods: Using standard autoantibody samples, the increased beat rate compared to the basal rate [delta beats/min] was recorded when investigating guideline required assay performance parameters.

Results: The 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.

Conclusion: This bioassay fulfilled guideline determined quality requirements and proved to be appropriate for its application in clinical trials.

Keywords: Biological sciences; Cardiology; Cell biology; Health sciences; Immunology; Medicine.

Figures

Fig. 1
Fig. 1
Example of the measurement of chronotropic effects of beta1-AAB on spontaneously beating neonatal rat cardiomyocytes with the IonOptix system.

References

    1. Wallukat G., Morwinski M., Kowal K., Förster A., Boewer V., Wollenberger A. Autoantibodies against the beta-adrenergic receptor in human myocarditis and dilated cardiomyopathy: beta-adrenergic agonism without desensitization. Eur. Heart J. 1991;12(Suppl. D):178–181.
    1. Matsui S., Fu M., Hayase M., Katsuda S., Yamaguchi N., Teraoka K., Kurihara T., Takekoshi N. Transfer of rabbit autoimmune cardiomyopathy into severe combined immunodeficiency mice. J. Cardiovasc. Pharmacol. 2003;42(Suppl. 1):S99–103.
    1. Jahns R., Boivin V., Hein L., Triebel S., Angermann C.E., Ertl G., Lohse M.J. Direct evidence for a beta 1-adrenergic receptor-directed autoimmune attack as a cause of idiopathic dilated cardiomyopathy. J. Clin. Invest. 2004;113:1419–1429.
    1. Patel P.A., Hernandez A.F. Targeting anti-beta-1-adrenergic receptor antibodies for dilated cardiomyopathy. Eur. J. Heart Fail. 2013;15:724–729.
    1. Wallukat G., Schimke I. Agonistic autoantibodies directed against G-protein-coupled receptors and their relationship to cardiovascular diseases. Semin. Immunopathol. 2014;36:351–363.
    1. Bornholz B., Roggenbuck D., Jahns R., Boege F. Diagnostic and therapeutic aspects of β1-adrenergic receptor autoantibodies in human heart disease. Autoimmun. Rev. 2014;13:954–962.
    1. Jahns R., Boege F. Questionable validity of peptide-based ELISA strategies in the diagnostics of cardiopathogenic autoantibodies that activate G-protein-coupled receptors. Cardiology. 2015;131:149–150.
    1. Sterin-Borda L., Cossio P.M., Gimeno M.F., Gimeno A.L., Diez C., Laguens R.P., Meckert P.C., Arana R.M. Effect of chagasic sera on the rat isolated atrial preparation: immunological, morphological and function aspects. Cardiovasc. Res. 1976;10:613–622.
    1. Wallukat G., Wollenberger A. Effects of the serum gamma globulin fraction of patients with allergic asthma and dilated cardiomyopathy on chronotropic beta adrenoceptor function in cultured neonatal rat heart myocytes. Biomed. Biochim. Acta. 1987;46:S634–S639.
    1. Nikolaev V.O., Boivin V., Störk S., Angermann C.E., Ertl G., Lohse M.J., Jahns R. A novel fluorescence method for the rapid detection of functional beta1-adrenergic receptor autoantibodies in heart failure. J. Am. Coll. Cardiol. 2007;50:423–431.
    1. Li H., Yu X., Liles C., Khan M., Vanderlinde-Wood M., Galloway A., Zillner C., Benbrook A., Reim S., Collier D., Hill M.A., Raj S.R., Okamoto L.E., Cunningham M.W., Aston C.E., Kem D.C. Autoimmune basis for postural tachycardia syndrome. J. Am. Heart Assoc. 2014;3:e000755.
    1. Fedorowski A., Li H., Yu X., Koelsch K.A., Harris V.M., Liles C., Murphy T.A., Quadri S.M.S., Scofield R.H., Sutton R., Melander O., Kem D.C. Antiadrenergic autoimmunity in postural tachycardia syndrome. Europace. 2017;19:1211–1219.
    1. Joshi-Barr S., Haberland A., Bartel S., Müller J., Choi T., Wallukat G. High throughput bioassay for beta1-adrenoceptor autoantibody detection. Int. J. Cardiol. 2016;219:98–104.
    1. Bornholz B., Benninghaus T., Reinke Y., Felix S.B., Roggenbuck D., Jahns-Boivin V., Jahns R., Boege F. 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. Clin. Chem. Lab. Med. 2016;54:683–691.
    1. Bornholz B., Hanzen B., Reinke Y., Felix S.B., Jahns R., Schimke I., Wallukat G., Boege F. Detection of DCM-associated β1-adrenergic receptor autoantibodies requires functional readouts or native human β1-receptors as targets. Int. J. Cardiol. 2016;202:728–730.
    1. Müller J., Wallukat G., Dandel M., Bieda H., Brandes K., Spiegelsberger S., Nissen E., Kunze R., Hetzer R. Immunoglobulin adsorption in patients with idiopathic dilated cardiomyopathy. Circulation. 2000;101:385–391.
    1. Dandel M., Wallukat G., Englert A., Lehmkuhl H.B., Knosalla C., Hetzer R. Long-term benefits of immunoadsorption in β(1)-adrenoceptor autoantibody-positive transplant candidates with dilated cardiomyopathy. Eur. J. Heart Fail. 2012;14:1374–1388.
    1. Guidance for Industry, Bioanalytical Method Validation, May 2001, U.S. Department of Health and Human Services, FDA, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), (accessed May 19, 2016).
    1. Guideline Bioanalytical method validation - WC500109686.pdf, EMEA/CHMP/EWP/192217/2009 Rev.1 Corr.2**, February 2012, (accessed September 2, 2016).
    1. Haberland A., Wallukat G., Dahmen C., Kage A., Schimke I. Aptamer neutralization of beta1-adrenoceptor autoantibodies isolated from patients with cardiomyopathies. Circ. Res. 2011;109:986–992.
    1. G. Brendelberger, CONCEPT HEIDELBERG, Validation of Analytical Methods: Intermediate Precision, ECA Academy, GMP News 13, December 13, 2001, (accessed May 18, 2016).
    1. Wallukat G., Muñoz Saravia S.G., Haberland A., Bartel S., Araujo R., Valda G., Duchen D., Diaz Ramirez I., Borges A.C., Schimke I. Distinct patterns of autoantibodies against G-protein-coupled receptors in Chagas’ cardiomyopathy and megacolon. Their potential impact for early risk assessment in asymptomatic Chagas’ patients. J. Am. Coll. Cardiol. 2010;55:463–468.
    1. Diana T., Li Y., Olivo P.D., Lackner K.J., Kim H., Kanitz M., Kahaly G.J. Analytical performance and validation of a bioassay for thyroid-blocking antibodies. Thyroid Off. J. Am. Thyroid Assoc. 2016;26:734–740.

Source: PubMed

Sponsors et collaborateurs

Les conditions médicales

Interventions en matière de drogue

3
S'abonner