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

Stoll, Anja (2007): Purification and Partial Characterization of Canine Neutrophil Elastase and the Development of an Immunoassay for the Measurement of Neutrophil Elastase Concentration in Serum. Dissertation, LMU München: Faculty of Veterinary Medicine

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 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.

• Item Type: Theses (Dissertation, LMU Munich)
• Keywords: Protein purification, neutrophil elastase, ELISA
• Subjects: 500 Natural sciences and mathematics > 590 Zoological sciences; 500 Natural sciences and mathematics
• Faculties: Faculty of Veterinary Medicine
• Language: English
• Date of oral examination: 9. February 2007
• 1. Referee: Hirschberger, Johannes
• Signature of the printed copy: 0001/UMC 16232