Analytical validation of an immunoassay for the quantification of N-terminal pro–B-type natriuretic peptide in feline blood

The measurement of N-terminal pro-B-type natriuretic peptide (NT-proBNP), a biomarker for heart stress detectable in blood, has been shown to have clinical utility in cats with heart disease. A second-generation feline enzyme-linked immunosorbent assay (Cardiopet® proBNP, IDEXX Laboratories Inc., Westbrook, Maine) was developed to measure NT-proBNP in routine feline plasma or serum samples with improved analyte stability. Results of the analytical validation for the second-generation assay are presented. Analytic sensitivity was 10 pmol/l. Accuracy of 103.5% was determined via serial dilutions of 6 plasma samples. Coefficients of variation for intra-assay, interassay, and total precision were in the ranges of 1.6-6.3%, 4.3-8.8%, and 10.1-15.1%, respectively. Repeatability across 2 lots for both serum and plasma had an average coefficient of determination (r(2)) of 0.99 and slope of 1.11. Stability of the analyte was found to be high. In serum samples held at 4°C for 24-72 hr, the mean percent recovery from time zero was ≥99%. In serum samples held at 25°C for 24 hr, the mean percent recovery from time zero was 91.9%, and for 48 hr, 85.6%. A method comparison of the first- and second-generation assays with a clinically characterized population of cats revealed no difference in the tests' ability to differentiate levels of NT-proBNP between normal cats and cats with occult cardiomyopathy (P < 0.001). Results from our study validate that the second-generation feline Cardiopet proBNP assay can measure NT-proBNP in routine feline plasma and serum samples with accuracy and precision.