Liu S, Zhang M Y, Song Q, Ling M, Kadijevic L, Shi Q
Spectral Diagnostics, Inc., Toronto, ON, Canada.
Clin Lab. 2001;47(1-2):19-27.
There has been a need to create stable and reproducible calibration and control materials for cardiac troponin I assays. Free troponin I, native or recombinant, has been known to be unstable, while troponin CI complex can be easily dissociated in low concentrations or in the presence of chelating agents. In order to overcome these difficulties, two single chain troponin I-C polypeptides have been engineered and expressed separately in Escherichia coli. One consists of a full-length of human cardiac troponin I and C, termed as ScTnI-C and the other consists of a stable fragment (aa28-110) of human cardiac troponin I and a full-length troponin C, termed as ScTnI-C-2. Both ScTnI-C and ScTnI-C-2 were purified to homogeneity by affinity chromatography using anti-cTnI monoclonal antibodies. ScTnI-C and ScTnI-C-2 have apparent molecular weights of 45 kD and 30 kD by SDS-PAGE, respectively. Stability studies by Stratus showed that ScTn I-C and ScTnI-C-2 were stable for 4 months at 2-8 degrees C and at least one year at -20 degrees C. When incubated in human serum at 37 degrees C, ScTnI-C-2 was more resistant to proteolysis than ScTnI-C. ScTnI-C can be recognized by all commercial TnI immunoassays with excellent activity. ScTnI-C-2 can be recognized by all immunoassays that target the stable region of cardiac troponin I. Judging by their performances, ScTnI-C and ScTnI-C-2 are both superior materials to be used as calibrators and controls in clinical laboratories.
一直以来都需要为心肌肌钙蛋白I检测创建稳定且可重复的校准和对照材料。已知游离的天然或重组肌钙蛋白I不稳定，而肌钙蛋白C-I复合物在低浓度或存在螯合剂的情况下容易解离。为了克服这些困难，人们设计并在大肠杆菌中分别表达了两种单链肌钙蛋白I-C多肽。一种由全长人心脏肌钙蛋白I和C组成，称为ScTnI-C，另一种由人心脏肌钙蛋白I的稳定片段（第28-110位氨基酸）和全长肌钙蛋白C组成，称为ScTnI-C-2。使用抗cTnI单克隆抗体通过亲和色谱法将ScTnI-C和ScTnI-C-2都纯化至同质。通过SDS-PAGE分析，ScTnI-C和ScTnI-C-2的表观分子量分别为45 kD和30 kD。Stratus进行的稳定性研究表明，ScTn I-C和ScTnI-C-2在2-8℃下可稳定保存4个月，在-20℃下至少可稳定保存一年。当在37℃的人血清中孵育时，ScTnI-C-2比ScTnI-C更耐蛋白水解。ScTnI-C能被所有商业化的TnI免疫测定法识别，活性优异。ScTnI-C-2能被所有针对心脏肌钙蛋白I稳定区域的免疫测定法识别。从它们的性能来看，ScTnI-C和ScTnI-C-2都是临床实验室用作校准物和对照的优质材料。
