An immunoprecipitation coupled with fluorescent Western blot analysis for the characterization of a model secondary serum cardiac troponin I reference material.
BACKGROUND
Cardiac troponin I (cTnI) is considered the 'gold standard' cardiac biomarker. However, the result comparability of commercial cTnI immunoassays is still lacking despite the availability of NIST Standard Reference Material, SRM 2921 (human cardiac troponin). To facilitate the standardization of the cTnI immunoassays, a secondary reference material consisting of a panel of three cTnI-positive human serum pools is proposed by the IFCC Working Group on Standardization of Troponin I. The objective of this study is to develop measurement procedures for the characterization of the future secondary reference material using a pooled cTnI-positive serum sample as a development model.


METHODS
We used magnetic beads coupled with 6 different anti-cTnI monoclonal antibodies that bind specifically to different amino acid sequence regions of the cTnI molecule to immunoprecipitate cTnI proteins from the pooled cTnI-positive serum sample followed by sensitive detection using a fluorescent Western blot.


RESULTS
The degradation of cTnI in the pooled sample was detected and the concentration of cTnI was determined.


CONCLUSIONS
We demonstrated the utility of this measurement procedure in support of the development of the proposed secondary cTnI-positive, serum-based reference material.
He
Hua-Jun
HJ
Chemical Science and Technology Laboratory, National Institute of Standards and Technology (NIST), Gaithersburg, MD 20899-8312, USA. hua.he@nist.gov
Lowenthal
Mark S
MS
Cole
Kenneth D
KD
Bunk
David
D
Wang
Lili
L
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001774:Blood Chemical Analysis / Q000379:methods / Q000592:standards*; D015153:Blotting, Western / Q000379:methods*; D002417:Cattle; D006801:Humans; D007118:Immunoassay / Q000379:methods / Q000592:standards*; D047468:Immunoprecipitation / Q000379:methods*; D008163:Luminescent Measurements; D009206:Myocardium / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D012015:Reference Standards; D013050:Spectrometry, Fluorescence / Q000379:methods*; D019210:Troponin I / Q000097:blood* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D019210:Troponin I