Enzyme-linked immunosorbent assay for canine α1-protease inhibitor

Abstract

Objective - To develop and validate an ELISA for quantifying α1-protease inhibitor (α1-PI) in serum and fecal extracts. Procedure - Affinity-purified rabbit origin canine α1-PI antibodies were biotinylated and, after addition of streptavidin-horseradish peroxidase, were used as the labeled complex in a noncompetitive immunoassay. The α1-PI standards were made from purified serum canine α1-PI diluted in phosphate-buffered saline solution containing 5% newborn calf serum and 0.01% thimerosal. This assay was validated by determining linearity, recovery of added α1-PI, detection limit, and intra- and interassay precision. Control range for serum and fecal α1-PI concentration was determined for samples from 25 healthy pet dogs. Results - The standard curve was linear between 5 and 50 ng/ml. Curves plotted after serial dilution of serum also were linear and ran parallel to the standard curve. Between- and within-assay coefficients of variation were 9.1 and 8.7%, respectively. The accuracy of the assay was tested by measuring the recovery of α1-PI added to serum and fecal extracts and ranged from 93 to 111%. The reference range of fecal α1-PI concentration was 0.023 to 5.67 (mean ± SD, 2.0 ± 1.82) μg/g and of serum α1-PI was 0.901 to 1.96 (1.42 ± 0.32) g/L. Conclusions - This ELISA had high precision, accuracy, and reproducibility for quantification of canine α1-PI concentration in serum and fecal extracts. Clinical Relevance - Specific ELISA for α1-PI may be a useful test for the diagnosis of protein-losing enteropathy in dogs, and as such, will facilitate further characterization and better understanding of this canine disorder.