BACKGROUND: Cardiac troponin I (cTnI) is the current standard biomarker for diagnosing acute myocardial infarction and for risk-stratification of acute coronary syndromes in patients. However, it remains unclear how the epitope specificity of antibodies in immunoassays influences the detection of various modified forms of cTnI. METHODS: Four mouse anti-human cTnI monoclonal antibodies targeting different regions of human cTnI were chosen for immunoaffinity purification of cTnI from human and swine cardiac tissue. High-resolution intact protein mass spectrometry was employed to assess the comparative performance of these four antibodies in detecting modified forms of cTnI. RESULTS: Our data revealed that antibody selection significantly impacts the relative protein yield of cTn from immunoaffinity purification. Remarkably, a single amino acid variation in cTnI (G->S) in the epitope region completely abolished the binding between monoclonal antibody 560 and swine cTnI in solution. Moreover, proteolytic degradation around the epitope region severely compromised the detection of proteolytic fragment forms of cTnI by monoclonal antibodies. In contrast, the phosphorylation status near the epitope region did not significantly affect the antibody recognition of cTnI. CONCLUSION: Caution needs to be taken in the interpretation of the data produced by immuno-assays with monoclonal antibodies against various epitopes of cTnI.
BACKGROUND:Cardiac troponin I (cTnI) is the current standard biomarker for diagnosing acute myocardial infarction and for risk-stratification of acute coronary syndromes in patients. However, it remains unclear how the epitope specificity of antibodies in immunoassays influences the detection of various modified forms of cTnI. METHODS: Four mouse anti-humancTnI monoclonal antibodies targeting different regions of humancTnI were chosen for immunoaffinity purification of cTnI from human and swine cardiac tissue. High-resolution intact protein mass spectrometry was employed to assess the comparative performance of these four antibodies in detecting modified forms of cTnI. RESULTS: Our data revealed that antibody selection significantly impacts the relative protein yield of cTn from immunoaffinity purification. Remarkably, a single amino acid variation in cTnI (G->S) in the epitope region completely abolished the binding between monoclonal antibody 560 and swinecTnI in solution. Moreover, proteolytic degradation around the epitope region severely compromised the detection of proteolytic fragment forms of cTnI by monoclonal antibodies. In contrast, the phosphorylation status near the epitope region did not significantly affect the antibody recognition of cTnI. CONCLUSION: Caution needs to be taken in the interpretation of the data produced by immuno-assays with monoclonal antibodies against various epitopes of cTnI.
Authors: E M Antman; M J Tanasijevic; B Thompson; M Schactman; C H McCabe; C P Cannon; G A Fischer; A Y Fung; C Thompson; D Wybenga; E Braunwald Journal: N Engl J Med Date: 1996-10-31 Impact factor: 91.245
Authors: A G Katrukha; A V Bereznikova; V L Filatov; T V Esakova; O V Kolosova; K Pettersson; T Lövgren; T V Bulargina; I R Trifonov; N A Gratsiansky; K Pulkki; L M Voipio-Pulkki; N B Gusev Journal: Clin Chem Date: 1998-12 Impact factor: 8.327
Authors: Ziqing Lin; Fang Guo; Zachery R Gregorich; Ruixiang Sun; Han Zhang; Yang Hu; Dhanansayan Shanmuganayagam; Ying Ge Journal: J Am Soc Mass Spectrom Date: 2018-04-09 Impact factor: 3.109