Negative Interference in Cardiac Troponin I Immunoassays by Circulating Troponin Autoantibodies

Abstract

Background: There are numerous potential sources of interference in immunoassays. Our aim was to identify the blood component that causes negative interference in cardiac troponin I (cTnI) immunoassays based on antibodies against the central part of cTnI. Methods: We isolated an interfering factor (IF) from a sample with low recovery of added cTnI, using several consecutive purification steps: caprylic acid precipitation, ammonium sulfate precipitation, and purification on Cibacron Blue gel and protein G columns. Purified IF was identified by gel electrophoresis and mass spectrometric analysis of protein bands. For the direct detection of human antibodies to cardiac troponin in serum samples, we developed immunoassays using three different anti-human immunoglobulin antibodies and measured troponin antibodies in samples with low and normal cTnI recovery. Results: Treatment with caprylic acid did not precipitate IF, but IF precipitated at 40% ammonium sulfate saturation. IF bound to a Cibacron Blue gel column, from which it was eluted with a linear salt gradient; it also bound to protein G. Gel electrophoresis of purified IF showed two major bands with molecular masses corresponding to the heavy (ϳ50 kDa) and light chains (ϳ25 kDa) of immunoglobulin, and their identities were confirmed by mass spectrometry. The presence of troponin-specific autoantibodies was confirmed in samples with low recoveries of cTnI by three different immunoassays. The median signals were significantly

National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines: Use of Cardiac Troponin and B-Type Natriuretic Peptide or N-Terminal proB-Type Natriuretic Peptide for Etiologies Other than Acute Coronary Syndromes and Heart Failure

“…Although the incidence of assay interferences caused by atypical antibodies has been reduced, all immunoassays have the potential for both false-positive and false-negative interference (23,24 ).…”

High incidence of macrotroponin I with a high-sensitivity troponin I assay

“…Historically, autoantibodies to cTnI have caused negative interference in some cTnI immunoassays [24,25]. Investigation of the epitope specificity of cTnI autoantibodies has shown that the cTnI midfragment (amino acids 30-100) is the commonest target of these antibodies [26,27].…”

Critical role of RAGE and HMGB1 in inflammatory heart disease

“…Recent work from our laboratory and others has focused on the dysregulation of the immune system as an essential modulator of disease induction and progression in heart failure (1,2). In this context, release of cardiac troponin I (TnI) from damaged cardiomyocytes into the circulation is believed to trigger an autoimmune response to TnI (3,4).…”