ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR CANINE ALPHA(1)-PROTEASE INHIBITOR

Objective-To develop and validate an ELISA for quantifying alpha(1)-pr otease inhibitor (alpha(1)-PI) in serum and fecal extracts. Procedure- Affinity-purified rabbit origin canine alpha(1)-PI antibodies were bio tinylated and, after addition of streptavidin-horseradish peroxidase, were used as the labeled complex in a noncompetitive immunoassay. The alpha(1)-PI standards were made from purified serum canine alpha(1)-PI diluted in phosphate-buffered saline solution containing 5% newborn c alf serum and 0.01% thimerosal. This assay was validated by determinin g linearity, recovery of added alpha(1)-PI, detection limit, and intra -and interassay precision. Control range for serum and fecal alpha(1)- PI concentration was determined for samples from 25 healthy pet dogs. Results-The standard curve was linear between 5 and 50 ng/ml. Curves p lotted after serial dilution of serum also were linear and ran paralle l to the standard curve. Between-and within-assay coefficients of vari ation were 9.1 and 8.7%, respectively. The accuracy of the assay was t ested by measuring the recovery of alpha(1)-PI added to serum and feca l extracts and ranged from 93 to 111%. The reference range of fecal al pha(1)-PI concentration was 0.023 to 5.67 (mean +/- SD, 2.0 +/- 1.82) mu g/g and of serum alpha(1)-PI was 0.901 to 1.96 (1.42 +/- 0.32) g/L. Conclusions-This ELISA had high precision, accuracy, and reproducibil ity for quantification of canine alpha(1)-PI concentration in serum an d fecal extracts.Clinical Relevance-Specific ELISA for alpha(1)-PI may be a useful test for the diagnosis of protein-losing enteropathy in d ogs, and as such, will facilitate further characterization and better understanding of this canine disorder.