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Purification and partial characterization of canine neutrophil elastase and the development of an immunoassay for the measurement of canine neutrophil elastase in serum obtained from dogs

Anja Stoll Dr med vet1, Jan S. Suchodolski Dr med vet, PhD2, Craig G. Ruaux BVSc, PhD3, and Jörg M. Steiner Dr med vet, PhD4
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  • 1 Gastrointestinal Laboratory, Department of Small Animal Clinical Science, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4474.
  • | 2 Gastrointestinal Laboratory, Department of Small Animal Clinical Science, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4474.
  • | 3 Gastrointestinal Laboratory, Department of Small Animal Clinical Science, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4474.
  • | 4 Gastrointestinal Laboratory, Department of Small Animal Clinical Science, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4474.

Abstract

Objective—To purify neutrophil elastase (NE) from dog blood and develop and validate an ELISA for the measurement of canine NE (cNE) in canine serum as a marker for gastrointestinal tract inflammation.

Sample Population—Neutrophils from 6 dogs immediately after they were euthanatized and serum from 54 healthy dogs.

Procedures—cNE was purified from blood by use of dextran sedimentation, repeated cycles of freezing-thawing and sonication, cation-exchange chromatography, and continuous elution electrophoresis. Antibodies against cNE were generated in rabbits, and an ELISA was developed and validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and interassay variability. A reference range was established by assaying serum samples from the 54 healthy dogs and by use of the lower 97.5th percentile.

Results—cNE was successfully purified from blood, and antibodies were successfully generated in rabbits. An ELISA was developed with a sensitivity of 1,100 μg/L. The reference range was established as < 2,239 μg/L. Ratios of observed-to-expected results for dilutional parallelism for 4 serum samples ranged from 85.4% to 123.1%. Accuracy, as determined by spiking recovery, ranged from 27.1% to 114.0%. Coefficient of variation for 4 serum samples was 14.2%, 16.0%, 16.8%, and 13.4%, respectively, for intra-assay variability and 15.4%, 15.0%, 10.5%, and 14.6%, respectively, for interassay variability.

Conclusions and Clinical Relevance—The purification protocol used here resulted in rapid and reproducible purification of cNE with a high yield. The novel ELISA yielded linear results and was accurate and precise. Additional studies are needed to evaluate the clinical usefulness of this assay.

Abstract

Objective—To purify neutrophil elastase (NE) from dog blood and develop and validate an ELISA for the measurement of canine NE (cNE) in canine serum as a marker for gastrointestinal tract inflammation.

Sample Population—Neutrophils from 6 dogs immediately after they were euthanatized and serum from 54 healthy dogs.

Procedures—cNE was purified from blood by use of dextran sedimentation, repeated cycles of freezing-thawing and sonication, cation-exchange chromatography, and continuous elution electrophoresis. Antibodies against cNE were generated in rabbits, and an ELISA was developed and validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and interassay variability. A reference range was established by assaying serum samples from the 54 healthy dogs and by use of the lower 97.5th percentile.

Results—cNE was successfully purified from blood, and antibodies were successfully generated in rabbits. An ELISA was developed with a sensitivity of 1,100 μg/L. The reference range was established as < 2,239 μg/L. Ratios of observed-to-expected results for dilutional parallelism for 4 serum samples ranged from 85.4% to 123.1%. Accuracy, as determined by spiking recovery, ranged from 27.1% to 114.0%. Coefficient of variation for 4 serum samples was 14.2%, 16.0%, 16.8%, and 13.4%, respectively, for intra-assay variability and 15.4%, 15.0%, 10.5%, and 14.6%, respectively, for interassay variability.

Conclusions and Clinical Relevance—The purification protocol used here resulted in rapid and reproducible purification of cNE with a high yield. The novel ELISA yielded linear results and was accurate and precise. Additional studies are needed to evaluate the clinical usefulness of this assay.

Contributor Notes

Presented in part at the 2006 Annual Forum of the American College of Veterinary Internal Medicine, Louisville, May 2006.

Address correspondence to Dr. Stoll.