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Development of a qualitative and quantitative detection method for the N-terminal fragment of cardiac myosin-binding protein C

Session Poster Session 5

Speaker Athiththan Yogeswaran

Event : ESC Congress 2018

  • Topic : basic science
  • Sub-topic : Basic Science - Cardiac Diseases: Biomarkers
  • Session type : Poster Session

Authors : A Yogeswaran (Giessen,DE), C Lipps (Giessen,DE), T Keller (Bad Nauheim,DE), C Liebetrau (Bad Nauheim,DE), O Doerr (Giessen,DE), S Wolter (Bad Nauheim,DE), S Voss (Bad Nauheim,DE), S Kriechbaum (Bad Nauheim,DE), M Aslam (Giessen,DE), S Sadayappan (Cincinnati,US), C W Hamm (Giessen,DE), C Troidl (Bad Nauheim,DE)

Authors:
A. Yogeswaran1 , C. Lipps1 , T. Keller2 , C. Liebetrau2 , O. Doerr3 , S. Wolter2 , S. Voss2 , S. Kriechbaum2 , M. Aslam1 , S. Sadayappan4 , C.W. Hamm3 , C. Troidl2 , 1Justus-Liebig University of Giessen, Experimental Cardiology - Giessen - Germany , 2Kerckhoff Heart and Thorax Center, Department of Cardiology - Bad Nauheim - Germany , 3University Hospital Giessen and Marburg, Department of Angiology and Cardiology - Giessen - Germany , 4University of Cincinnati, Internal Medicine - Cincinnati - United States of America ,

Citation:
European Heart Journal ( 2018 ) 39 ( Supplement ), 996-997

Background: Cardiac myosin-binding protein C (MYBPC3) was recently discovered as a new biomarker for acute coronary syndrome. Due to the active and early cleavage of MYBPC3, the N-terminal fragment C0-C1f is released within the first minutes of ischemia and during the early reperfusion phase. Even though C0-C1f has been proposed to be a novel early cardiac biomarker, there is no known specific detection method for this peptide.

Purpose: Our aim is to establish a detection method for qualitative and quantitative detection of C0-C1f. The core benefit of this assay will be the evaluation of C0-C1f as a novel cardiac biomarker for non-invasive differential diagnosis of non-ST-elevation myocardial infarction.

Methods: An indirect sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of C0-C1f and the full-length protein MYBPC3. Using a monoclonal mouse antibody against the N-terminus of MYBPC3 developed for this assay, the parameters of the ELISA were optimized to obtain a sensitive detection method. Its specificity was confirmed by western blotting. Prefixed depletion using a magnetic bead-conjugated antibody against the intermediary region of MYBPC3 was applied to remove analogue epitopes that differ from the target C0-C1f (e.g. C0-C2 and full-length). The efficiency of this method was confirmed via mass spectrometry and ELISA.

Results: Cross-reactivity of the antibodies used regarding slow and fast skeletal myosin-binding protein C and mouse isoforms was determined and specificity for the cardiac isoform was verified. The ELISA is precise and sensitive with a lower limit of detection of 0.05 ng/mL and its characteristics, such as dilution-linearity and recovery values correspond to the accepted standards. MYBPC3 plasma levels of healthy individuals (n=4) are 10 ng/mL respectively.

Conclusions: This assay is the first method developed for measuring the concentration of C0-C1f. It is suitable for qualitative and quantitative detection of C0-C1f. This method may aid in evaluating the role of C0-C1f in progression and development of cardiac diseases. As a next step, the concentration of C0-C1f in different cardiac diseases, including acute coronary syndrome, and its release kinetics will be determined to evaluate its utility as a novel cardiac biomarker

Assay characteristics
TypeSandwich ELISA
Detection methodFluorescent
ReactivityHuman, Mouse
SpecifityCardiac Myosin-binding Protein C; No significant cross-reactivity or interference between MYBPC3 and analogs was observed
Lower Limit of Detection50 ng/L

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