Detection by ELISA of C-terminal proBNP in plasma from cats with cardiomyopathy

The B-type natriuretic peptide prohormone (proBNP) is enzymatically cleaved into an inactive N-terminal peptide and a biologically active C-terminal peptide with many beneficial cardiorenal effects. The purpose of this study was to develop and test in cats with cardiomyopathy an immunoassay to quantify the concentrations of C-terminal proBNP in feline plasma. An anti-canine proBNP monoclonal antibody (UI-1021) was shown to have adequate binding affinity to proBNP 80-106 for use in a solid-phase immunoassay, and by epitope mapping to bind within positions 84-87 of feline proBNP. UI-1021 was paired with an affinity-purified rabbit polyclonal detection antibody to feline proBNP 100-106, in a sandwich ELISA with feline proBNP 80-106 standard. The linearity and analytical range and sensitivity of the assay were confirmed from 1.4 to 85 pmol/L. Spike recovery averaged 106.5% (95% confidence interval 78-135%). Within run and intra-assay coefficients of variation were <12%. A protease inhibitor mixture preserved proBNP 80-106 immunoreactivity for at least 5 days in plasma. Clinical verification of the ELISA was done using plasma from 13 cats with cardiomyopathy, whose C-terminal proBNP concentrations ranged from 1.7 to 78.8 pmol/L vs. <1.4-1.8 pmol/L in plasma from 18 healthy cats. Concentrations were found to be substantially lower than reported N-terminal proBNP concentrations, and similar to those of human heart failure patients where relative C-terminal BNP deficiencies have been proposed as contributory to the progression of the disease.