Development of a Qualitative Sequential Immunoassay for Characterizing the Intrinsic Properties of Circulating Cardiac Troponin I

Lin, Yixin; Fu, Qin; Zhu, Jie; Miller, Julie M.; Eyk, Jennifer E. Van

doi:10.1373/clinchem.2009.135186

Cited by 11 publications

(12 citation statements)

References 36 publications

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“…Currently, the comprehensive analysis of cTnI variants under these approaches requires multiple assays and multiple preparation protocols. Our laboratory has developed a quantitative cTnI assay using one capture antibody and multiple detection antibodies with distinct epitopes, thus quantifying 5 different epitopes simultaneously in a single sequential assay [18]. This approach can be adapted to the determination and quantification of cTnI along with its degradation and phosphorylated variants, as well as cTnI bound in its troponin complex.…”

Section: Prospects Of Clinical Ctni Post Translational Modification Amentioning

confidence: 99%

“…Our efforts to translate these findings to a clinically relevant assay have unfortunately been hampered by limitations inherent to analytical technologies. Although we have tested many platforms [13,18,19] we are only now approaching the technical sensitivity and specificity required for quantitative analysis of total circulating cTnI, with routine analysis of modified amino acids on the horizon and the simultaneous analysis of total and modified cTnI further still.…”

Section: Introductionmentioning

confidence: 99%

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The continuing evolution of cardiac troponin I biomarker analysis: from protein to proteoform

Soetkamp

Raedschelders

Mastali

et al. 2017

Expert Review of Proteomics

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Introduction: The troponin complex consists of three proteins that fundamentally couple excitation with contraction. Circulating cardiac-specific Troponin I (cTnI) serves as diagnostic biomarker tools for risk stratification of acute coronary syndromes and acute myocardial infarction (MI). Within the heart, cTnI oscillates between inactive and active conformations to either block or disinhibit actinomyosin formation. This molecular mechanism is fine-tuned through extensive protein modifications whose profiles are maladaptively altered with co-morbidities including hypertrophic cardiomyopathy, diabetes, and heart failure. Technological advances in analytical platforms over the last decade enable routine baseline cTnI analysis in patients without cardiovascular complications, and hold potential to expand cTnI readouts that include modified cTnI proteoforms. Areas covered: This review covers the current state, advances, and prospects of analytical platforms that now enable routine baseline cTnI analysis in patients. In parallel, improved mass spectrometry instrumentation and workflows already reveal an array of modified cTnI proteoforms with promising diagnostic implications. Expert commentary: New analytical capabilities provide clinicians and researchers with an opportunity to address important questions surrounding circulating cTnI in the improved diagnosis of specific patient cohorts. These techniques also hold considerable promise for new predictive and prescriptive applications for individualized profiling and improve patient care.

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Section: Prospects Of Clinical Ctni Post Translational Modification Amentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The continuing evolution of cardiac troponin I biomarker analysis: from protein to proteoform

Soetkamp

Raedschelders

Mastali

et al. 2017

Expert Review of Proteomics

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“…As a result, the capture antibody was attached to the functionalized waveguide using biotin-avidin chemistry. Antibodies attached to magnetic beads or other substrates have also been reported previously (Hideshima et al, 2008;Lin et al, 2010;Mukundan et al, 2012;Ohk and Bhunia, 2013;Zhou et al, 2011).…”

mentioning

confidence: 79%

“…The interaction between avidin and biotin has been exploited to conjugate antibodies on a solid surface and has been widely used in the immobilization process (Iversen et al, 2008;Lin et al, 2006Lin et al, , 2010Orth et al, 2003). Biotinylated antibodies react with avidin and other biotin-binding proteins, including streptavidin, neutravidin, tamavidin, and captavidin (Garcia-Aljaro et al, 2009), to generate a biocompatible layer on a surface by exploiting one of the strongest bonds (K d D 10 15 M ¡1 ) (Airenne et al, 1999;Diamandis and Christopoulos, 1991).…”

Section: Antibody Immobilization Based On Avidin-biotin Reactionmentioning

confidence: 99%

Technological Development of Antibody Immobilization for Optical Immunoassays: Progress and Prospects

Wang

et al. 2014

Critical Reviews in Analytical Chemistry

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The high sensitivity of optical detection techniques and the highly specific reactions between antibodies and antigens mean that optical immunoassays have attracted much interest in the fields of protein, hormone, drug, and microorganism detection, without the need for complex separation and extraction steps. The immobilization of an antibody on a solid support is a crucial step for optical immunoassays. This review surveys the latest advances in current antibody immobilization techniques in detail, including physical adsorption, covalent attachment, bioaffinity immobilization, and some recently developed methods. Furthermore, specific consideration is given to oriented immobilization, which may improve the homogeneous surface covering the accessibility of the active site and surface coverage, and the analytical performance of immunoassays. Finally, new perspectives for antibody immobilization techniques in optical immunoassays are outlined.

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“…Taken together, our results suggest that caution needs to be taken in the interpretation of data produced by immunoassays with mAbs against different epitopes of cTnI. Arguably it may become necessary to utilize sequential immunoassays involving multiple antibodies for capture and detection of cTnI in its various modified forms and complexes [7,34]. …”

Section: Introductionmentioning

confidence: 99%

The impact of antibody selection on the detection of cardiac troponin I

Guy

Chen

Clinton

et al. 2013

Clinica Chimica Acta

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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.

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Development of a Qualitative Sequential Immunoassay for Characterizing the Intrinsic Properties of Circulating Cardiac Troponin I

Cited by 11 publications

References 36 publications

The continuing evolution of cardiac troponin I biomarker analysis: from protein to proteoform

The continuing evolution of cardiac troponin I biomarker analysis: from protein to proteoform

Technological Development of Antibody Immobilization for Optical Immunoassays: Progress and Prospects

The impact of antibody selection on the detection of cardiac troponin I

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