Analytical validation of a second-generation immunoassay for the quantification of N-terminal pro-B-type natriuretic peptide in canine blood.

N-terminal pro-B-type natriuretic peptide (NT-proBNP) has been shown to have clinical utility as a biomarker in dogs with heart disease. There were several limitations associated with early diagnostic assay formats including a limited dynamic range and the need for protease inhibitors to maintain sample stability. A second-generation Cardiopet® proBNP enzyme-linked immunosorbent assay (IDEXX Laboratories Inc., Westbrook, Maine) was developed to address these limitations, and the present study reports the results of the analytical method validation for the second-generation assay. Coefficients of variation for intra-assay, interassay, and total precision based on 8 samples ranged from 3.9% to 8.9%, 2.0% to 5.0%, and 5.5% to 10.6%, respectively. Analytical sensitivity was established at 102 pmol/l. Accuracy averaged 102.0% based on the serial dilutions of 5 high-dose canine samples. Bilirubin, lipids, and hemoglobin had no effect on results. Reproducibility across 3 unique assay lots was excellent with an average coefficient of determination (r (2)) of 0.99 and slope of 1.03. Both ethylenediamine tetra-acetic acid plasma and serum gave equivalent results at time of blood draw (slope = 1.02, r (2) = 0.89; n = 51) but NT-proBNP was more stable in plasma at 25°C with median half-life measured at 244 hr and 136 hr for plasma and serum, respectively. Plasma is the preferred sample type and is considered stable up to 48 hr at room temperature whereas serum should be frozen or refrigerated when submitted for testing. Results of this study validate the second-generation canine Cardiopet proBNP assay for accurate and precise measurement of NT-proBNP in routine sample types from canine patients.

Performance validation and method comparison of an in-clinic enzyme-linked immunosorbent assay for the detection of canine pancreatic lipase.

Diagnosis of pancreatitis is often difficult in dogs that present with acute vomiting, anorexia, and abdominal pain, as these clinical signs may occur with a variety of other illnesses. While quantitative reference laboratory methods specific for canine pancreatic lipase are available to aid in diagnosis, results are generally not available until the next day. The objective of the current study was to validate a semiquantitative in-clinic rapid test for the measurement of canine pancreas-specific lipase (cPL) and to compare its performance to the reference lab method. Comparison of the reference method for cPL to the in-clinic assay demonstrated 96-100% agreement for canine serum samples with normal levels of cPL and 88-92% agreement for samples with elevated levels of cPL. Common interfering substances such as bilirubin, lipids, or hemoglobin had no effect on assay performance. Both within-day and day-to-day variations ranged from 10% to 20% of the calculated cPL concentration, which demonstrated a high degree of precision for the in-clinic assay. Performance of 3 lots of the in-clinic assay with the same set of canine serum samples demonstrated high assay reproducibility, with interclass correlation coefficients of ≥0.93. Results of the in-clinic cPL assay, based on both visual and calculated cPL concentrations, were consistent throughout 15 months of storage. The in-clinic test provides immediate, semiquantitative results to supplement existing pancreatitis diagnostics at the time of acute illness. Because the reference and in-clinic methods are aligned, they can be used together as an immediate aid pet-side and as a fully quantitative follow-up test at the reference laboratory.

A role for extracellular and transmembrane domains of Sef in Sef-mediated inhibition of FGF signaling.

Sef (similar expression to fgf genes) is a member of the fibroblast growth factor (FGF) synexpression group that negatively regulates FGF receptor (FGFR) signaling in zebrafish during early embryonic development and in mammalian cell culture systems. The mechanism by which Sef exerts its inhibitory effect remains controversial. It has been reported that Sef functions either through binding to and inhibiting FGFR1 activation or by acting downstream of FGF receptors at the level of MEK/ERK kinases. In both cases, the intracellular domain of Sef was found to play a role in the inhibitory function of Sef. Here we demonstrated that both extracellular and transmembrane domains of Sef contributed to Sef-mediated negative regulation of FGF signaling. In fact, over-expression studies in NIH3T3 cells showed that a truncated mutant of Sef, which lacks the intracellular domain (SefECTM), exerted the inhibitory activity on FGF signaling by inhibiting FGFR1 tyrosine phosphorylation and subsequent activation of the Raf/MEK/ERK signaling cascade. We also showed that SefECTM associated with FGFR1, and inhibited FGF-induced ERK activation in HEK293T cells. Furthermore, we demonstrated that the over-expression of SefECTM was able to inhibit the function of a constitutively activated form of FGFR1, FGFR1-C289R, but not FGFR1-K562E. Finally, we found that SefECTM reduced cell viability when over-expressed in human umbilical vein endothelial cells (HUVEC). These data provide additional insight into the structure-activity relationship in the mechanism of inhibitory action of Sef on FGFR1-mediated signaling.