Development and validation of a sandwich ELISA for measurement of angiotensinogen concentration in canine urine and serum

Objective To develop and analytically validate a sandwich ELISA that measures angiotensinogen concentration in canine urine and serum samples. Methods Serum (n = 4) and urine (10) samples submitted to a clinical pathology laboratory were used for assay development. Recombinant canine angiotensinogen and both rabbit-derived polyclonal and mouse-derived monoclonal antibodies against it were obtained from a commercial source. A sandwich ELISA was developed and validated via assessment of precision, limit of blank, dilutional linearity, recovery, and cross-reactivity with albumin and angiotensin II. Results Intra- and interassay variability ranged from 8.31% to 14.52% and 1.88% to 16.87%, respectively. The limit of blank of the assay was 1.01 ng/mL, which was lower than all observed concentrations in 4 serum (27,130 to 64,690 ng/mL) and 10 urine (3.24 to 8,970.24 ng/mL) samples. Both serum and urine demonstrated ideal dilutional linearity following 2-fold serial dilutions ( R 2 > 0.99). The assay showed moderate recovery (61.8% to 113.9%). Minimal cross-reactivity was observed with albumin and angiotensin II, as both yielded values below the assay's limit of blank. Conclusions The angiotensinogen ELISA we developed is precise, linear, and sufficiently sensitive for measuring angiotensinogen in the urine and serum of domestic dogs. Clinical Relevance Urinary angiotensinogen quantification is a promising noninvasive marker of intrarenal renin-angiotensin system activity in humans and rodents; this assay enables evaluation of its utility in dogs.