Longitudinal studies of cardiac troponin-I concentrations in serum from male Sprague Dawley rats: baseline reference ranges and effects of handling and placebo dosing on biological variability.

Serum cardiac troponin-I has been validated as a biomarker for cardiotoxicity in numerous animal models; however, baseline reference ranges for cTnI concentration in a healthy population of laboratory rats, as well as an investigation of biological cTnI variability in rats with respect to time, handling, and placebo dosing methods, have not been reported. In this study, we used an ultrasensitive cTnI immunoassay to quantify hourly concentrations of cTnI in live rats handled under standard laboratory conditions using 15 microL of serum per determination. The baseline reference range (mean 4.94 pg/mL, range 1-15 pg/mL, 99% confidence interval [CI]) of cTnI concentration in rats was consistent with previously reported reference ranges for cTnI in humans (1-12 pg/mL) and with preliminary studies in dogs (1-4 pg/mL) and monkeys (4-5 pg/mL) using the same cTnI assay method. In addition, cTnI concentrations in individual rat serum samples show minimal biological variability over a twenty-four-hour interval when compared to a meaningful reference change value of 193% to 206%. Furthermore, measurements of cTnI concentration were consistent within the reference limits in individual rats over long periods and under three different standard laboratory handling conditions. Thus, using this new method, rats can be followed longitudinally at hourly intervals, and a doubling of cTnI concentration would be significant above biological variability. This is a new paradigm for preclinical testing, which allows transient changes in cTnI concentration to be accurately quantified. This understanding of baseline and biological variability in rats will be fundamental for designing and analyzing future studies that assess potential cardiotoxicity in drug development.

Analytical validation of a highly sensitive microparticle-based immunoassay for the quantitation of IL-13 in human serum using the Erenna immunoassay system.

IL-13 is a Th2 cytokine that has been shown to be an important mediator of airway inflammation contributing to asthma lesions. Given its proposed role in asthma, measurements of this cytokine in serum may provide insights into disease mechanisms, progression and pharmacodynamic effects of IL-13 targeted therapeutics. However, current commercially available ELISA immunoassays are frequently unable to detect baseline concentrations of IL-13 in serum from healthy individuals, which are below the limit of detection. Here we describe the use of the novel microparticle-based Erenna IL-13 human immunoassay (Singulex, Inc.), which utilizes proprietary antibodies and single molecule counting technology, to quantify IL-13 from 100 microL of serum from apparently healthy subjects and clinically defined symptomatic and asymptomatic asthma subjects. The lower limit of quantification of the Erenna assay was validated at 0.07 pg/mL and the assay detected baseline concentrations of IL-13 in 98% of serum samples tested. The calibration curve showed good precision over the entire linear range of 0.07-50 pg/mL, with inter-assay imprecision <10% CV except at the lowest concentration tested (<15%). The intra- and inter-assay imprecision of spiked serum samples containing three different IL-13 concentrations (2, 8, and 25 pg/mL) ranged from 2.2-2.4% and 6.1-6.8%, respectively. Using the Erenna IL-13 assay, we observe that serum IL-13 concentrations range from <0.07-1.02 pg/mL in apparently healthy subjects (N=60) with similar ranges in asymptomatic (0.07-0.66 pg/mL, N=26) and symptomatic (<0.07-1.26 pg/mL, N=96) asthma subjects. The Erenna immunoassay improved sensitivity by over two full logs compared to previous ELISA methods, while using smaller sample volumes. In addition, the Erenna assay reliably measured IL-13 in endogenous and spiked human serum samples that were not quantifiable using other methods. Taken together, these results show that this novel assay offers a significant improvement over previous methods for high-sensitive quantitative measurement of IL-13 in human serum samples obtained from both apparently healthy and asthmatic subjects, and can be used in future clinical studies to accurately measure concentrations of this cytokine prior to and following drug therapy in human serum.