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

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 mug/L. The reference range was established as < 2,239 mug/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.