Establishment and Application of a Dual-Labeling Time-Resolved Fluorescence Immunoassay Method for Simultaneous Detection of the Troponin I-C Complex and Full-Size-Troponin I

Biao Huang; Jian Wu; Hao Chen; Li Zhang; Xiumei Zhou; Qingqing Wu; Ting Li; Yigang Wang; Penguo Xia; Yaping Dai; Guoyin Kai; Pengfei Liu; Hao Pei

doi:10.3389/fcvm.2020.596051

Establishment and Application of a Dual-Labeling Time-Resolved Fluorescence Immunoassay Method for Simultaneous Detection of the Troponin I-C Complex and Full-Size-Troponin I

Front Cardiovasc Med. 2021 Jan 14:7:596051. doi: 10.3389/fcvm.2020.596051. eCollection 2020.

Authors

Biao Huang¹, Jian Wu^{2

3}, Hao Chen¹, Li Zhang¹, Xiumei Zhou¹, Qingqing Wu¹, Ting Li¹, Yigang Wang¹, Penguo Xia¹, Yaping Dai⁴, Guoyin Kai⁵, Pengfei Liu⁶, Hao Pei⁴

Affiliations

¹ Immounoassay Laboratory, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, China.
² State Key Laboratory for the Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.
³ Department of Laboratory Medicine, The First People's Hospital of Yancheng City, Yancheng, China.
⁴ Clinical Lab, Wuxi No.5 People's Hospital, Wuxi, China.
⁵ Chinese Medicine Resources Teaching and Research Laboratory, College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, China.
⁶ Department of Gastroenterology, The Jiangyin Clinical College of Xuzhou Medical University, Jiangyin, China.

Abstract

Background: The measurement of cardiac troponin I (cTnI) is widely used in the diagnosis of acute myocardial infarction (AMI). Although existing cTnI detection methods measure total cTnI, the significance of undegraded full-size-cTnI levels is still not well-understood. In this study, we have established a novel dual-labeling time-resolved fluorescence immunoassay (TRFIA) technique that simultaneously detects the cTnI-C complex and full-size-cTnI, allowing us to explore the clinical value of full-size-cTnI determination. Methods: An antibody against the 23-43 amino acid region of cTnI protected by endogenous cTnC is coupled to magnetic beads to provide a solid-phase antibody for capturing all cTnI. An antibody against cTnC in the cTnI-C complex labeled with Eu³⁺ was used to detect the cTnI-C complex, and an antibody labeled with Sm³⁺ near the C-terminal 190-203 amino acids of cTnI was used to detect full-size-cTnI. Through dual-labeling TRFIA, cTnI-C complex, full-size-cTnI, and the full-size-cTnI/cTnI-C ratio can be detected simultaneously. The dual-labeling TRFIA technique was used to analyze serum samples collected at different times during treatment and compare their full-size-cTnI/cTnI-C ratios. Results: The sensitivity for the cTnI-C-TRFIA complex was 0.02 ng/mL, the measurement range was 0.02-40 ng/mL, the average intra-batch coefficient of variation (CV) was 4.35%, and the inter-average CV was 6.23%. The correlation coefficient between cTnI-C-TRFIA and commercial cTnI-CLIA methods was R ² = 0.8887. The sensitivity for full-size-cTnI-TRFIA was 0.04 ng/mL, the measurement range was 0.04-40 ng/mL, the average intra-batch CV was 4.95%, and the average inter-batch CV was 7.79%. The correlation coefficient between full-size-cTnI-TRFIA and commercial cTnI-CLIA methods was R ² = 0.7247. Conclusions: Dual-labeling full-size-cTnI/cTnI-C-TRFIA analysis is helpful for determining the length of time of chest pain before admission and the degree of continuous release of cTnI in the myocardium. Thus, it is more for early prognosis than just detecting cTnI.

Keywords: acute myocardial infarction; cardiac troponin I; dual-labeled; immunoassay; time-resolved fluorescence.