Characteristics of myoglobin, carbonic anhydrase III and the myoglobin/carbonic anhydrase III ratio in trauma, exercise, and myocardial infarction patients.

Carbonic anhydrase III (CA III) is an enzyme present in skeletal muscle which is released into circulation following injury. Myoglobin (Mb) is a heme protein located in skeletal, smooth, and cardiac muscle which is also released after injury. Because CA III is not present in myocardium, combining serum CA III and Mb measurements may improve the specificity of Mb as an early diagnostic marker for myocardial infarction (MI) provided that a fixed ratio of Mb and CA III is released from skeletal muscle following cell injury. We examined release of Mb and CA III for exercise subjects (n=12), trauma patients (n=18), and MI patients (n=10) following emergency department admission. A fixed ratio of Mb/CA III had medians of 3.505 (range: 1.05-6.76) and 2.890 (range: 0.97-3.97) for exercise and trauma subjects, respectively, in samples collected within 5 h of the event. The Mb/CA III ratio was significantly higher (P<0.001) in MI patients (median: 35.395; range: 8.65-170.45) during this same time. This study confirmed that Mb and CA III are released in a fixed ratio following exercise, showed no significant difference in the ratio for trauma patients, and demonstrated significant ratio elevation for MI patients. These data suggest the ratio to be a useful diagnostic indicator of MI.

Removal of endotoxin from recombinant protein preparations.

OBJECTIVES: To develop an effective method to remove endotoxin from large scale E. coli recombinant protein purifications. DESIGN AND METHODS: Triton X-114 phase separation, affinity chromatography utilizing immobilized polymyxin B or immobilized histidine, were used to remove endotoxin from purified preparations of recombinant CK-BB, CK-MB, CK-MM, myoglobin, and cardiac troponin I. Endotoxin levels were measured by a Limulus Amebocyte Lysate gel-clot assay. The immunoactivity of these protein preparations was determined by BIAcore analysis using a panel of in-house generated monoclonal antibodies and by a Stratus Fluorometric Analyzer. In the case of troponin I, the BIAcore was also utilized to measure troponin C interactions. RESULTS: Phase separation with Triton X-114 was the most effective method in reducing the amount of endotoxin present in the protein preparations compared to either polymyxin B or histidine affinity chromatography. With Triton X-114, the reduction in endotoxin levels was greater than 99% and recovery of the proteins after endotoxin removal was greater than 90%. All three procedures for removing endotoxin had no deleterious effects on the immunoactivity of majority proteins when tested with a panel of monoclonal antibodies. Troponin I also retained its ability to bind to troponin C in the presence of Ca2+. Recombinant CK-BB and CK-MM which were expressed in the soluble fraction of E. coli cell lysates, contained significantly higher endotoxin levels than recombinant CK-MB, myoglobin and cardiac troponin I which were expressed in the form of inclusion bodies. CONCLUSION: Of the three methods tested, Triton X-114 phase separation was the most effective way of removing endotoxin from recombinant proteins.

Analytical performance and clinical utility of a sensitive immunoassay for determination of human cardiac troponin I.

OBJECTIVES: To determine the serum and plasma level of human cardiac troponin I (cTnI) resulting from myocardial damage, we have developed a sensitive and specific one-step enzyme immunoassay to measure cardiac troponin I. DESIGN AND METHODS: The COBAS cTnI assay is a semi-automated one-step solid phase immunoassay compatible with the COBAS Core. The assay is performed in a sandwich type format using a polyclonal goat antibody capture and two highly specific horseradish peroxidase conjugated monoclonal antibody detectors directed against different epitopes of the cTnI molecule. Calibrators were made with purified recombinant cTnI. RESULTS: The level of cTnI was determined in 84 healthy donors with no evidence of myocardial injury, resulting in a lower limit of detection (LLD) of 0.09 microgram/L. The upper reference limit (URL) of the normal reference range was calculated as 0.20 microgram/L. The dynamic range of the consequent EIA was between 0.09 and 6.0 micrograms/L with a total assay time of 45 min. Intra-assay and inter-assay variances (CVs) were < or = 4%. Cross-reactivity with fast and slow skeletal troponin I was absent in concentrations up to 2.0 mg/L. Common interferents yielded negative results in the cTnI assay. Clinical utility was confirmed by measuring the circulating serum or plasma levels of cardiac troponin I in serial samples from marathon runners, clinical samples from trauma patients, and patients presenting to the Emergency Department with complaints of chest pain. Results were further evaluated using clinical diagnosis at discharge and quantified concentrations of other cardiac markers by a Stratus analyzer and ELISA procedures. CONCLUSIONS: Results from normal and clinical samples assayed in house for cTnI concentrations indicate that the Spectral EIA is a highly sensitive means of quantifying cTnI levels in serum and plasma for acute cardiac syndrome. The cardiac specificity of cTnI over other well-known cardiac markers is reflected in experimental results and parallel clinical diagnosis.