Development and analytical validation of a radioimmunoassay for the measurement of alpha₁-proteinase inhibitor concentrations in feces from puppies and healthy adult dogs

Abstract

Canine α1-proteinase inhibitor (cα₁-PI), a proteolysis-resistant protein with a molecular weight similar to albumin, has been shown to be clinically useful as a marker for gastrointestinal protein loss in dogs. A competitive, liquid-phase radioimmunoassay was developed and analytically validated. Fecal samples were collected from 101 healthy pet dogs of various breeds and ages, and fecal cα₁-PI (Fcα₁-PI) concentrations were compared between dogs of different age groups. A reference interval for Fcα₁-PI concentration was calculated using the central 95th percentile. Analytical sensitivity of the assay was 2.2 μg Fcα₁-PI/g feces. Observed-to-expected ratios for the serial dilution and spiking recovery of 9 and 6 fecal extracts ranged from 90.4 to 152.0% and from 71.3 to 112.3%, respectively. Coefficients of variation for intra- and interassay variability for 6 fecal extracts were ≤10.8% and ≤12.5%, respectively. The reference intervals for the mean and maximum Fcα₁-PI from fecal samples collected on 3 consecutive days were 2.2 13.9 μg/g and 2.2-21.0 μg/g, respectively. Fcα₁-PI was significantly higher in dogs <1 year of age (P < 0.0001 for both mean and maximum Fcα₁-PI for the 3 samples). The radioimmunoassay described is sensitive, linear, precise, reproducible, and accurate for clinical use, thus allowing reliable quantification of Fcα₁-PI in clinical patients. Using this assay, a mean or a maximum Fc₁-PI for 3 sampling days of >13.9 μg/g or >21.0 μg/g, respectively, should be considered abnormal in dogs >1 year of age. Fecal cα₁ PI concentrations in dogs <1 year of age were significantly higher and should be carefully interpreted in this age group.