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Cross-laboratory analytical validation of the cardiac biomarker NT-proANP in rat
Petra Vinken1, William J Reagan2, Luis A Rodriguez3
1Janssen Research & Development, Janssen Pharmaceutica NV, Beerse, Belgium.
Introduction:
Natriuretic peptides, including N-terminal-proatrial natriuretic peptide (NT-proANP) are cardiac hormones that are produced in response to myocardial stretch and have been used in rats and humans as blood based functional cardiac biomarkers. There are limited validation data of these assays in rats and therefore the Predictive Safety Testing Consortium, Cardiac Hypertrophy Working Group (PSTC-CHWG) performed a cross-laboratory (5 laboratories) analytical evaluation of a commercially available NT-proANP ELISA for use with rat samples.
Methods:
Serum samples were collected from normal Sprague Dawley (SD) rats and were spiked with kit calibrator material or rat heart tissue extracts to provide specimens for the validation. In addition, the cardiotoxicant, isoproterenol, was used to induce elevated endogenous NT-proANP levels in a subgroup of rats for additional validation specimens. The Biomedica™ (BI-20892, Vienna, Austria) proANP (1-98) enzyme-linked immunoabsorbent assay (ELISA) kit was used to measure NT-proANP. Intra-assay and inter-assay precisions, accuracy, sample linearity, recovery, limit of detection, upper and lower limits of quantitation (ULOQ and LLOQ, respectively), sample-freeze/thaw stability and stored sample stability were assessed and compared to pre-determined acceptance criteria.
Results:
The majority of the experimental assessments met the established validation criteria, however there were individual results that did not meet these standards. Overall, acceptable intra- and inter-assay precisions and accuracies as well as inter-laboratory precision and accuracy were demonstrated. Linearity and recovery values fell within the pre-determined acceptance criteria, samples remained stable for up to three freeze-thaw cycles and frozen samples were stable at ~-70 °C for 12 months. The limit of detection (LOD) and LLOQ and ULOQ were similar to those specified by the manufacturer.
Discussion:
Overall, the assay was demonstrated to be technically adequate for the detection of NT-proANP serum levels in SD rats.
