Development and validation of a sandwich ELISA for measurement of angiotensinogen in feline urine

Objective: To develop a sandwich ELISA that measures angiotensinogen concentration in urine samples from domestic cats. Methods: Recombinant feline angiotensinogen and antibodies against it were generated by a commercial source. Angiotensinogen protein sequence was confirmed through tandem mass spectrometry. A sandwich ELISA was developed and validated for precision, limit of quantification, dilutional linearity, recovery, and crossreactivity with feline albumin and angiotensin II. Fresh voided urine samples collected from purpose-bred cats (n = 3) and residual urine samples from client-owned cats submitted to a diagnostic clinical pathology laboratory (3) were used for assay development and validation. Results: The assay showed good performance for measuring urinary angiotensinogen within a concentration range of 0.74 to 50 ng/mL. Intra- and interassay coefficients of variation were 8.7% to 18.5% and 19.4% to 29.0%, respectively. Dilutional linearity following 2-time serial dilutions was ideal (R2>0.98), and there was a moderate recovery rate (102.7% to 137.7%). Minimal crossreactivity was observed for albumin and angiotensin II. Urinary angiotensinogen concentrations ranged from 6.03 to 514.24 ng/mL in the samples evaluated using this ELISA. Conclusions: The sandwich ELISA developed offers sufficient sensitivity and minimal crossreactivity for measuring urinary angiotensinogen in cats. Assay precision based on coefficient of variation percentage was deemed acceptable given the magnitude of concentration differences observed between different samples; the assay is not well suited to detect subtle differences in angiotensinogen concentration. Clinical Relevance: Measuring urinary angiotensinogen is an established noninvasive method to evaluate intrarenal renin-angiotensin system activity in humans and rodents. This assay allows for the investigation of this method in cats.