Recombinant single chain cardiac troponin I-C polypeptides: superior calibration and control materials for cardiac troponin I immunoassays.
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.
Liu
S
S
Spectral Diagnostics, Inc., Toronto, ON, Canada.
Zhang
M Y
MY
Song
Q
Q
Ling
M
M
Kadijevic
L
L
Shi
Q
Q
D000911:Antibodies, Monoclonal; D002138:Calibration; D002846:Chromatography, Affinity; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007118:Immunoassay; D009206:Myocardium / Q000737:chemistry*; D010446:Peptide Fragments; D011786:Quality Control; D011994:Recombinant Proteins / Q000302:isolation & purification; D015203:Reproducibility of Results; D019209:Troponin C / Q000235:genetics; D019210:Troponin I / Q000097:blood* / Q000235:genetics
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019209:Troponin C; D019210:Troponin I