Purification and Partial Characterization of Canine Neutrophil Elastase and the Development of an Immunoassay for the Measurement of Neutrophil Elastase Concentration in Serum

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 euthanized dogs
and serum from 54 healthy dogs. Procedures—cNE was purified from
dog 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 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 g/L. The reference range was established as
< 2,239 g/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%. The 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 ELISA developed yielded
linear results and was accurate and precise. Additional studies are
needed to evaluate the clinical usefulness of this assay.