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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Editorial Board Member: Gillery, Philippe / Kazmierczak, Steven / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Schlattmann, Peter / Whitfield, John B.

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Development of a candidate secondary reference procedure (immunoassay based measurement procedure of higher metrological order) for cardiac troponin I: I. Antibody characterization and preliminary validation

James E. Noble1 / David M. Bunk2 / Robert H. Christenson3 / Kenneth D. Cole4 / Hua-Jun He4 / Alexei G. Katrukha5 / Mauro Panteghini6 / Robert A. Porter1 / Heinz Schimmel7 / Jillian R. Tate8 / Lili Wang4 /

1Analytical Science Group, National Physical Laboratory, Teddington, UK

2Chemical Science and Technology Laboratory, National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA

3Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, USA

4Division of Biochemical Science, National Institute of Standards and Technology, Gaithersburg, MD, USA

5HyTest Ltd., Turku, Finland

6Center for Metrological Traceability in Laboratory Medicine (CIRME), University of Milan, Milan, Italy

7European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, European Commission, Geel, Belgium

8Pathology Queensland, Department of Chemical Pathology, Royal Brisbane and Women's Hospital, Herston, Queensland, Australia

Corresponding authors: James E. Noble, Analytical Science Group, National Physical Laboratory, Teddington, UK Lili Wang, NIST, 100 Bureau Drive MS 8392, Gaithersburg, MD 20899-8392, USA Fax: +44 301 977-0685,

Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 48, Issue 11, Pages 1603–1610, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2010.316, November 2010

Abstract

In this study, the first steps in the development of a secondary reference measurement procedure (RMP) ‘higher metrological order measurement procedure’ to support the cardiac troponin I (cTnI) standardization initiative is described. The RMP should be used to assign values to serum-based secondary reference materials (RMs) without analytical artifacts causing bias. A multiplexed bead-based assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the optimum monoclonal antibody pair (clones 560 and 19C7) for the RMP. Using these antibodies, an ELISA-based procedure was developed to accurately measure the main cTnI forms present in blood. The proposed RMP appears to show no bias when tested on samples containing various troponin complexes, phosphorylated and dephosphorylated forms, and heparin. The candidate assay displayed suitable linearity and sensitivity (limit of detection, 0.052 μg/L) for the measurement of the proposed cTnI secondary RMs. Preliminary comparison data on patient samples with a commercial cTnI assay are also provided to support the suitability of RMP for value assignment to RMs. Full validation and final assessment of the RMP will be performed through transferability and inter-comparison studies.

Clin Chem Lab Med 2010;48:1603–10.

Keywords: antibody specificity; antigen-antibody reactions; candidate reference procedure; ELISA; reference standards; troponin I

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