Evaluation of recombinant enzyme calibration to harmonize lipoprotein-associated phospholipase A2 activity results between instruments.

OBJECTIVES: Enzymatic activity of lipoprotein-associated phospholipase A2 (Lp-PLA2) mediates vascular inflammation in coronary heart disease (CHD). Calibration of Lp-PLA2 activity measurements using a recombinant enzyme was performed to assess intra- and inter-laboratory assay precision and accuracy in routine clinical settings. DESIGN AND METHODS: Test performance assessment included recovery, analytical sensitivity, linear range, within-lab and site-to-site precision, interference, and analyte stability. Results using the Beckman-Coulter AU400 analyzer were compared to other chemistry analyzers. RESULTS: Lp-PLA2 activity ranged from 84 to 303nmol/min/mL in 300 subjects, with 82.0% and 18.0% measurements below and at or above a cut-point of 225nmol/min/mL, respectively. Results of matched K2-EDTA plasma and serum (n=131) were similar with a slope of 1.00, y-intercept of 0.05, and R-value of 0.988. Mean recovery ranged from 90 to 106% of baseline after storage at different temperatures and time periods. Limit of detection was ≤10nmol/min/mL, without deviation from linearity between 10 and 382nmol/min/mL. Endogenous substances and medications did not interfere with the activity measurements. Overall intra- and inter-laboratory precision among three sites showed coefficients of variation of ≤3.8% and ≤5% respectively. Limit of quantitation was 1.3nmol/min/mL. Method comparison studies for multiple analyzers demonstrated slopes, intercepts or R(2) coefficients ranging from 0.96 to 1.06, -5.6 to 2.0, or 0.997 to 0.999, respectively. CONCLUSION: Analytical performance of the calibrated PLAC(®) test for Lp-PLA2 enzyme activity assay in CHD is resistant to a wide variety of pre-analytical factors, with site-to-site reproducibility on multiple analyzers sufficient to standardize results in diverse laboratory settings.

A biosensor for theophylline based on fluorescence detection of ligand-induced hammerhead ribozyme cleavage.

Recently, Breaker and coworkers engineered hammerhead ribozymes that rearrange from a catalytically inactive to an active conformation upon allosteric binding of a specific ligand. To monitor cleavage activity in real time, we have coupled a donor-acceptor fluorophore pair to the termini of the substrate RNA of such a hammerhead ribozyme, modified to cleave in trans in the presence of the bronchodilator theophylline. In the intact substrate, the fluorophores interact by fluorescence resonance energy transfer (FRET). The specific FRET signal breaks down as the effector ligand binds, the substrate is cleaved, and the products dissociate, with a rate constant dependent on the concentration of the ligand. Our biosensor cleaves substrate at 0.46 min(-1) in 1 mM theophylline and 0.04 min(-1) without effector, and discriminates against caffeine, a structural relative of theophylline. We have measured the theophylline-dependence profile of this biosensor, showing that concentrations as low as 1 microM can be distinguished from background. To probe the mechanism of allosteric regulation, a single nucleotide in the communication domain between the catalytic and ligand-binding domains was mutated to destabilize the inactive conformation of the ribozyme. As predicted, this mutant shows the same activity (0.3 min(-1)) in the presence and absence of theophylline. Additionally, time-resolved FRET measurements on the biosensor ribozyme in complex with a noncleavable substrate analog reveal no significant changes in fluorophore distance distribution upon binding of effector.