title: The impact of antibody selection on the detection of cardiac troponin I.
abstract: 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.
  
  
  CONCLUSIONS
  Caution needs to be taken in the interpretation of the data produced by immuno-assays with monoclonal antibodies against various epitopes of cTnI.
authors.affiliation: Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53706, United States.
authors.forename: Moltu J
authors.initials: MJ
authors.lastname: Guy
authors.forename: Yi-Chen
authors.initials: YC
authors.lastname: Chen
authors.forename: Laura
authors.initials: L
authors.lastname: Clinton
authors.forename: Han
authors.initials: H
authors.lastname: Zhang
authors.forename: Jiang
authors.initials: J
authors.lastname: Zhang
authors.forename: Xintong
authors.initials: X
authors.lastname: Dong
authors.forename: Qingge
authors.initials: Q
authors.lastname: Xu
authors.forename: Serife
authors.initials: S
authors.lastname: Ayaz-Guner
authors.forename: Ying
authors.initials: Y
authors.lastname: Ge
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D007118:Immunoassay; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D013552:Swine; D019210:Troponin I / Q000032:analysis / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D019210:Troponin I