Longitudinal studies of cardiac troponin I concentrations in serum from male cynomolgus monkeys: resting values and effects of oral and intravenous dosing on biologic variability.

BACKGROUND: There is a paucity of information regarding cardiac troponin (cTn) concentrations in peripheral blood of nonhuman primates (NHP). Even less is known regarding cTn concentrations in monkeys that are restrained for oral or intravenous (iv) dosing. OBJECTIVES: The objectives of these studies were to (1) determine cardiac troponin I (cTnI) concentration in resting Cynomolgus monkeys and investigate biologic variability in cTnI concentration over time, (2) determine cTnI changes in restrained monkeys given sham oral dosing, and (3) determine cTnI changes in restrained NHP given a sham intravenous dosing. METHODS: The Research Use Only Erenna cTnI ultrasensitive immunoassay based on single molecule counting technology was used to determine serum cTnI concentration in longitudinal studies of male Cynomolgus monkeys at rest, and after sham oral and intravenous dosing. Animals were catheterized prestudy, and blood samples were collected by an automated sampling device to limit disturbance of the animals during studies. RESULTS: In resting monkeys cTnI concentrations were relatively low and constant and ranged from 0.2 to 9.6 pg/mL (mean = 2.5 pg/mL), with minimal variability during a 24-hour period. Animals given sham oral dosing also had low cTnI concentration with little variability similar to the resting values. Several animals restrained for intravenous dosing had a small transient increase in cTnI concentration (~5-25 pg/mL) that resolved quickly within one to 3 hours postinjection. CONCLUSIONS: Results of this longitudinal study provide information that may be important in differentiating effects of animal handling from those associated with compound-related effects in preclinical toxicology studies of drugs in development.

A highly sensitive novel immunoassay specifically detects low levels of soluble Aß oligomers in human cerebrospinal fluid.

INTRODUCTION: Amyloid ß-protein oligomers play a key role in Alzheimer's disease (AD), but well-validated assays that routinely detect them in cerebrospinal fluid (CSF) are just emerging. We sought to confirm and extend a recent study using the Singulex Erenna platform that reported increased mean CSF oligomer levels in AD. METHODS: We tested four antibody pairs and chose one pair that was particularly sensitive, using 1C22, our new oligomer-selective monoclonal antibody, for capture. We applied this new assay to extracts of human brain and CSF. RESULTS: A combination of 1C22 for capture and 3D6 for detection yielded an Erenna immunoassay with a lower limit of quantification of approximately 0.15 pg/ml that was highly selective for oligomers over monomers and detected a wide size-range of oligomers. Most CSFs we tested had detectable oligomer levels but with a large overlap between AD and controls and a trend for higher mean levels in mild cognitive impairment (MCI) than controls. CONCLUSION: Aß oligomers are detectable in most human CSFs, but AD and controls overlap. MCI CSFs may have a modest elevation in mean value by this assay.

A perspective on the use of iTRAQ reagent technology for protein complex and profiling studies.

Proteomic research includes the characterization of protein mixtures in order to understand complex biological systems and determine relationships between proteins, their function, and protein-protein interactions. Often the goal of such research is to monitor changes of proteins in perturbed systems, a type of study referred to as differential expression analysis. To perform these studies requires the ability to execute some type of differential comparison of a given protein state in reference to some type of a control. The iTRAQ reagents are a set of isobaric reagents which are amine specific and allow for the identification and quantitation of up to four different samples simultaneously. The amine specificity of these reagents makes most peptides in a sample amenable to this labeling strategy with no loss of information from samples involving post-translational modifications, such as the scrutiny of signal transduction pathways that often involve phosphorylation phenomena. In addition, the multiplexing capacity of these reagents allows for information replication within certain LC-MS/MS experimental regimes, providing additional statistical validation within any given experiment. The results presented herein demonstrate a few examples of the wide variety of quantitative information that can be realized when undertaking such experimental approaches. These include temporal analysis of drug-induced-protein expression, discovery and elucidation of disease markers, and protein-protein interactions in multi-protein complexes.