Accessible Requires Authentication Published by De Gruyter November 1, 2010

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. Noble, David M. Bunk, Robert H. Christenson, Kenneth D. Cole, Hua-Jun He, Alexei G. Katrukha, Mauro Panteghini, Robert A. Porter, Heinz Schimmel, Jillian R. Tate, Lili Wang and on behalf of the IFCC Working Group on Standardization of Troponin I (WG-TNI)

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.


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,

Published Online: 2010-11-01
Published in Print: 2010-11-01

©2010 by Walter de Gruyter Berlin New York