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.

Degradation of cardiac troponin I in serum complicates comparisons of cardiac troponin I assays.

BACKGROUND: Up to a 20-fold variation in serum cardiac troponin I (cTnI) concentration may be observed for a given patient sample with different analytical methods. Because more limited variation is seen for control materials and for purified cTnI, we explored the possibility that cTnI was present in altered forms in serum. METHODS: We used four recombinantly engineered cTnI fragments to study the regions of cTnI recognized by the Stratus(R), Opus(R), and ACCESS(R) immunoassays. The stability of these regions in serum was analyzed with Western blot. RESULTS: The measurement of several control materials and different forms of purified cTnI using selected commercial assays demonstrated five- to ninefold variation. Both the Stratus and Opus assays recognized the N-terminal portion (NTP) of cTnI, whereas the ACCESS assay recognized the C-terminal portion (CTP) of cTnI. Incubation of recombinant cTnI in normal human serum produced a marked decrease in cTnI concentration as determined with the ACCESS, but not the Stratus, immunoassay. Western blot analysis of the same samples using cTnI NTP- and CTP-specific antibodies demonstrated preferential degradation of the CTP of cTnI. CONCLUSIONS: The availability of serum cTnI epitopes is markedly affected by the extent of ligand degradation. The N-terminal half of the cTnI molecule was found to be the most stable region in human serum. Differential degradation of cTnI is a key factor in assay-to-assay variation.

An enzymatic method for oxalate automated with the Cobas Fara centrifugal analyzer.

Measurement of oxalate in urine has been automated for use with the Cobas Fara centrifugal analyzer. No sample pretreatment other than (a critical) pH adjustment is required. Between-run CVs were less than 4%. Results were linearly related to oxalate concentration to 1000 mumol/L. Ascorbic acid ingestion, up to 5 g of ascorbate daily, caused no demonstrable interference with the assay. This practical, automated method for assaying urinary oxalate is substantially faster than other chemical or enzymatic methods.

Screening of malignant hyperthermia susceptible families by creatine phosphokinase measurement and other clinical investigations.

We investigated 56 families afflicted with malignant hyperpyrexia. One hundred and twenty-four individuals within these families had had an episode of malignant hyperthermia, of whom we saw seventy-two. Serum creatine phosphokinase (CPK) was statistically higher in affected individuals and in close relatives than in normal volunteers. The magnitude of the serum CPK elevations varied significantly between families. While in some families the serum CPK was clearly elevated in affected individuals, in other families the serum CPK was normal or only moderately or inconsistently raised. In these latter families serum CPK measurement was therfore of little or no value in identifying afflicted members. The incidence of musculoskeletal abnormalities was greater in affected individuals and in close relatives than in the general population. Thus, the concomitant individual belonging to a family known to be susceptible to malignant hyperthermia was a better indicator of the MH trait than was the presence of only one of these parameters. For reasons which we do not fully understand, MHS individuals were found to require fewer anaesthetics than normal persons. The incidence of MH crises within each family fell significantly following investigation, counselling, and issuance of Medic-Alert bracelets.