Development of a qualitative sequential immunoassay for characterizing the intrinsic properties of circulating cardiac troponin I.
BACKGROUND
With myocardial infarction (MI), cardiac troponin is released from the heart into circulation, where it can be detected with immunoassays independently quantifying cardiac troponin I (cTnI) or cTnT. There is, however, no single immunoassay that sequentially probes the posttranslational modification status of cTnI or directly characterizes whether circulating cTnI is bound to cTnC and/or cTnT. Here we describe the development of a qualitative immunoassay to directly probe the primary and ternary structure of circulating cTnI through diffractive optics technology (dotLab System, Axela).


METHODS
Anti-cTnI antibody 8I-7 was immobilized on a patterned sensor to capture cTnI. One or more detector antibodies were sequentially introduced to probe for amino acid sequence integrity or phosphorylation status of cTnI, or its association with cTnC and/or cTnT. Respective immunocaptures were recorded as real-time diffractive intensities (DIs), and the DI differences were analyzed. Each immunodetection was independent of the others but was done in a single sequential assay.


RESULTS
This diffraction-based immunoassay successfully characterized cTnI. The unamplified assay determined whether cTnI was degraded at N-terminus and/or C-terminus or phosphorylated. Sequential application of multiple detector antibodies without an antibody-stripping step enables real-time interrogation of 5 different epitopes of cTnI, or direct detection of the cTn complex (cTnI-cTnC-cTnT) in a single sequential assay. Finally, this assay was optimized with amplification to directly detect circulating cTnI bound to cTnC and cTnT in serum from an MI patient.


CONCLUSIONS
The dot Immunoassay is the first qualitative sequential immunoassay to address the direct interactions of the troponin subunits and various modified forms of cTnI.
Lin
Yixin
Y
Axela Inc., Toronto, Ontario, Canada.
Fu
Qin
Q
Zhu
Jie
J
Miller
Julie M
JM
Van Eyk
Jennifer E
JE
D006801:Humans; D007118:Immunoassay; D046912:Multiprotein Complexes / Q000097:blood; D009203:Myocardial Infarction / Q000097:blood; D010766:Phosphorylation; D015203:Reproducibility of Results; D019209:Troponin C / Q000097:blood; D019210:Troponin I / Q000097:blood*; D020107:Troponin T / Q000097:blood
D046912:Multiprotein Complexes; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T