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We evaluated a new protocol for measurement of cyclosporine A (CsA) 2 H after dose (C2) on the V-Twin® analyzer. Imprecision, recovery, and linearity were determined using CsA-spiked blood pools. Accuracy was evaluated using specimens from renal, cardiac, and liver transplant patients, and results were compared with those from liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the Abbott TDx®/TDxFLx® assay. Cross-reactivity and interferences were assessed in the presence of 800 ng/mL CsA. Imprecision coefficients of variation were 3.3%-4.8% (within run) and 5.9%-8.7% (total). Recovery was within 10% of the expected values. Linearity was 350-2,000 ng/mL. Calibration was stable for ≥ 2 weeks. Method comparison showed regression statistics: V-Twin® = 1.01 × LC tandem MS + 36.1, r = 0.971; V-Twin® = 1.13 × Abbott - 92.4, r = 0.969. Metabolite cross-reactivity and interference (endogenous substances and drugs) were within ±10%. The C2 protocol on the V-Twin® analyzer provides acceptable assay performance and accurate determination of whole blood CsA drawn at 2 H after dose., (Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.)

Cardiac troponin I (cTnI) is the inhibitory subunit of the troponin complex and is a biochemical marker for myocardial infarction (MI). It is found in human serum within 4-6 h following MI. One of us has shown [Morjana (1998) Biotechnol. Appl. Biochem. 28, 105-111] that MI patient serum TnI is cleaved at the N- and C-terminals and that the TnI fragments exist as a complex with tropinin C (TnC) and troponin T (TnT). In the present study, we have generated C-terminal truncated TnI fragments and studied their immunological and biochemical properties. Human recombinant TnI (rTnI) expressed in Escherichia coli is cleaved into a major fragment with a molecular mass of 17500 Da using CNBr. The major CNBr fragment contains the first 153 amino acids of human cTnI (TnI153). Cleavage of the rTnI with the endoproteinase Asp-N generates a smaller TnI fragment (TnI88, residues 6-96). TnI153 has higher immunological activity than that of rTnI and lower activity than that of TnI88, as judged by the Stratus II TnI Immunoassay. TnI153 exhibits biochemical and immunological properties similar to those of intact TnI. It binds TnC at a molar ratio of 1:1 and forms a ternary complex with TnC and TnT. TnC enhances the immunological activity of TnI153, but has little effect on the activity of TnI88. The TnI153-TnC complex exhibits higher immunological activity than rTnI-TnC and TnI88-TnC, and much higher activity than free rTnI, TnI153 and TnI88. The presence of TnT has no effect on the immunological activity of the TnI153-TnC complex, suggesting that the addition of TnT does not interfere with TnI153 recognition by TnI monoclonal antibodies. Free TnI153 and TnI88 degrade rapidly in human serum. TnC protects TnI153 from proteolytic degradation, but offers less protection for TnI88. The TnI88-TnC complex lost 80% of its immunological activity after incubation for 2 days in human serum at 37 degrees C. However, there was no loss in the immunological activity of the TnI153-TnC complex under the same conditions. A cTnI fragment (TnI80, residues 1-80), expressed in E. coli as a fusion protein, exhibits immunological activity and stability similar to that of TnI88.

Cardiac troponin I (TnI) is the inhibitory subunit of the troponin complex and a specific biochemical marker for myocardial infarction (MI). It is released into the bloodstream within 4-6 h following MI, peaks after 18-24 h and remains elevated for up to 7 days. In this work, I have identified TnI forms present in MI-patient serum. By immobilizing anti-TnI antibodies, which recognize various epitopic sites on the TnI molecule, we were able to isolate TnI from MI-patient serum pools. Western-blot analysis following SDS/PAGE shows two major TnI fragments with apparent molecular masses of 18000 and 14000 Da. Either or both fragments are seen in serum obtained from individuals with MI. The fragments are generated as a result of proteolytic processing from the C-terminal region of TnI. Partial processing from the N-terminal of TnI is also seen and is associated with the generation of the 14000 Da fragment. Very little unprocessed intact TnI is detected in patient serum after MI. The degradation in vitro of cardiac TnI was studied by incubating either bovine or human recombinant TnI in serum. Western-blot analyses with TnI antibody showed that purified TnI spiked into normal human serum or MI-patient serum depleted of TnI degrades rapidly to lower molecular mass fragments. Degradation of TnI is associated with a loss in immunological activity. Serum TnI isolated by anti-TnI antibody, under non-dissociating conditions, is associated with at least troponin C (TnC) and troponin T (TnT). This complex is bound by anti-TnI, anti-TnC and anti-TnT antibodies.

Human cardiac troponin I has been expressed at high level in Escherichia coli as a fusion protein by using the expression vector Ptac114. The expressed protein forms primarily intracellular inclusion bodies that are solubilized in the presence of 8 M urea. The purified troponin I is recognized by anti-(human cardiac troponin I) monoclonal antibodies. Equilibrium denaturation of recombinant human troponin I and bovine troponin I is compared by monitoring changes in the protein's fluorescence and CD characteristics. At pH 7.5 the equilibrium denaturation of both proteins by urea occurs in two distinct steps involving at least three major conformational states: native, intermediate and fully denatured. The biphasic profile in the presence of urea is observed by both fluorescence and CD spectroscopy. In the intermediate state the native tertiary structure is largely disrupted and 40% of the secondary structure is conserved, as suggested by near-UV and far-UVCD respectively. Thermal denaturation of troponin I, as followed by fluorescence, shows a loss in the signal that is not reversible after heating to 90 degrees C. In the presence of a constant amount of urea (not greater than 0.5 M) the thermal denaturation becomes biphasic, suggesting the accumulation of an intermediate species that is stabilized by urea. The fluorescence of 1-anilino-8-naphthalenesulphonate produced on binding troponin I decreases in the presence of increasing concentrations of urea up to 3 M; at higher urea concentrations no further change in the remaining signal is observed. Kinetic studies show at least two phases of renaturation for troponin I previously denatured with 8 M urea, whereas only a single phase is detected for the renaturation process in the presence of 3 M urea. The results suggest the occurrence of a stable folding intermediate, the formation of which might be related to the two-domain architecture of troponin I.