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Enzyme-linked immunosorbent assay for canine alpha 1-protease inhibitor
T Melgarejo1, D A Williams, E K Asem
1Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907-1248, USA.
Objective:
To develop and validate an ELISA for quantifying alpha 1-protease inhibitor (alpha 1-PI) in serum and fecal extracts.
Procedure:
Affinity-purified rabbit origin canine alpha 1-PI antibodies were biotinylated 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 calf serum and 0.01% thimerosal. This assay was validated by determining 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 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 alpha 1-PI added to serum and fecal extracts and ranged from 93 to 111%. The reference range of fecal alpha 1-PI concentration was 0.023 to 5.67 (mean +/- SD, 2.0 +/- 1.82) micrograms/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 reproducibility for quantification of canine alpha 1-PI concentration in serum and fecal extracts.
Clinical Relevance:
Specific ELISA for alpha 1-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.