Enzyme-linked immunosorbent assay for canine alpha 1-protease inhibitor.
OBJECTIVE
To develop and validate an ELISA for quantifying alpha 1-protease inhibitor (alpha 1-PI) in serum and fecal extracts.


METHODS
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.


CONCLUSIONS
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.
Melgarejo
T
T
Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907-1248, USA.
Williams
D A
DA
Asem
E K
EK
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005243:Feces / Q000737:chemistry; D011817:Rabbits; D012015:Reference Standards; D015203:Reproducibility of Results; D000515:alpha 1-Antitrypsin / Q000032:analysis* / Q000302:isolation & purification
D000906:Antibodies; D000515:alpha 1-Antitrypsin