HyTest Ltd., Intelligate 1, 6th Floor, Joukahaisenkatu 6, 20520 Turku, Finland.
Department of Biochemistry, Faculty of Biology, Moscow State University, Leninskie Gory 1, str. 12, 119234 Moscow, Russia.
Int J Mol Sci. 2024 Aug 16;25(16):8919. doi: 10.3390/ijms25168919.
Immunodetection of cardiac isoforms of troponin I (cTnI) and troponin T (cTnT) in blood samples is widely used for the diagnosis of acute myocardial infarction. The cardiac troponin complex (ITC-complex), comprising cTnI, cTnT, and troponin C (TnC), makes up a large portion of troponins released into the bloodstream after the necrosis of cardiomyocytes. However, the stability of the ITC-complex has not been fully investigated. This study aimed to investigate the stability of the ITC-complex in blood samples. A native ITC-complex was incubated in buffer solutions, serum, and citrate, heparin, or EDTA plasma at various temperatures. Western blotting and gel filtration were performed, and troponins were detected using specific monoclonal antibodies. The ITC-complex dissociated at 37 °C in buffers with or without anticoagulants, in citrate, heparin, and EDTA plasmas, and in serum, into a binary cTnI-TnC complex (IC-complex) and free cTnT. In plasma containing heparin and EDTA, the IC-complex further dissociated into free TnC and cTnI. No dissociation was found at 4 °C or at room temperature (RT) in all matrices within 24 h except for EDTA plasma. After incubation at 37 °C in EDTA plasma and serum, dissociation was accompanied by proteolytic degradation of both cTnI and cTnT. The presence of anti-troponin autoantibodies in the sample impeded dissociation of the ITC-complex. The ITC-complex dissociates in vitro to form the IC-complex and free cTnT at 37 °C but is mostly stable at 4 °C or RT. Further dissociation of the IC-complex occurs at 37 °C in plasmas containing heparin and EDTA.
免疫检测血液样本中心脏肌钙蛋白 I（cTnI）和肌钙蛋白 T（cTnT）的同工型，广泛用于急性心肌梗死的诊断。肌钙蛋白复合物（ITC-复合物）由 cTnI、cTnT 和肌钙蛋白 C（TnC）组成，在心肌细胞坏死后，大量肌钙蛋白释放到血液中。然而，ITC-复合物的稳定性尚未得到充分研究。本研究旨在研究血液样本中 ITC-复合物的稳定性。将天然 ITC-复合物在不同温度下于缓冲液、血清和柠檬酸盐、肝素或 EDTA 血浆中孵育。进行 Western blot 和凝胶过滤，并使用特异性单克隆抗体检测肌钙蛋白。ITC-复合物在有或没有抗凝剂的缓冲液中、柠檬酸盐、肝素和 EDTA 血浆中以及血清中于 37°C 时解离，形成二元 cTnI-TnC 复合物（IC-复合物）和游离 cTnT。在含有肝素和 EDTA 的血浆中，IC-复合物进一步解离为游离 TnC 和 cTnI。在所有基质中，除 EDTA 血浆外，在 4°C 或室温（RT）下 24 小时内未发现解离。在 EDTA 血浆和血清中于 37°C 孵育时，解离伴随着 cTnI 和 cTnT 的蛋白水解降解。样品中存在抗肌钙蛋白自身抗体会阻碍 ITC-复合物的解离。ITC-复合物在体外于 37°C 时解离形成 IC-复合物和游离 cTnT，但在 4°C 或 RT 时主要稳定。在含有肝素和 EDTA 的血浆中，IC-复合物在 37°C 时进一步解离。
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