Analytical validation of a novel agglutination immunoassay for the quantification of cystatin B in canine and feline urine

Urinary cystatin B (uCysB) is a biomarker of kidney injury in dogs and cats. A high-throughput agglutination immunoassay (Idexx Laboratories) was developed for widespread commercial availability of uCysB testing in a reference laboratory setting. We evaluated immunoassay performance and included analyses of precision, accuracy, linearity, interference, analytical specificity, lot-to-lot variation, and stability. CVs from precision studies on the range of 50-500 ng/mL were 0.38-2.53% (canine) and 0.44-3.5% (feline) for within-run precision, and 1.49-5.09% (canine) and 0.65-5.05% (feline) for between-run precision. Accuracy was measured by recovery percentage and was 89-101% (canine) and 91-112% (feline). Amoxicillin, ciprofloxacin, low concentrations of doxycycline, bilirubin, glucose, ketones, RBCs, hemoglobin, cloudiness, lipids, protein, and pH did not affect results. Urinary cystatin A did not cross-react with the uCysB immunoassay. Results of lot-to-lot linear regressions were 0.90-1.07 (slopes) and 0.97-1.00 (coefficient of determination). One or more freeze-thaw cycles and storage at 30°C impacted the immunoassay stability of canine samples but not feline samples under the same conditions. Our results validate this novel agglutination immunoassay for accurate and precise measurement of uCysB in canine and feline urine samples. For optimal immunoassay performance, samples should be kept at 4°C for a maximum of 1 wk. Our uCysB immunoassay is a useful and practical tool to be used in assessing kidney injury in canine and feline patients in the clinical setting.