Development of an enzyme-linked immunosorbent assay for equine neutrophil elastase measurement in blood: preliminary application to colic cases.
Abstract: Equine neutrophil elastase (NE) is a protease released in inflammatory diseases and participating in tissue destruction. To measure NE in horse plasma to assess its role in pathological conditions, we purified elastase from equine neutrophils by a double step chromatography and obtained a pure protein of 27 kDa, 4 kDa smaller than the NE 2A previously purified (Scudamore et al., 1993; Dagleish et al., 1999), which was likely to be NE 2B. We developed an ELISA by using two specific polyclonal antibodies obtained from rabbit and guinea pig. The sandwich complex was detected using a secondary antibody conjugated to alkaline phosphatase. The ELISA showed good precision and accuracy, with intra- and inter-assay coefficients of variation below 10% for equine NE concentrations ranging from 1.875 to 60 ng/ml. A stable plasma NE value, unaffected by the delay of centrifugation (over 4h), was obtained with plasma from EDTA anticoagulated blood. The mean value (+/-SEM) measured in 37 healthy horses was 32.53+/-4.6 ng/ml. NE level in plasma of horses with colic at the time of admission was significantly higher than in healthy horses. Our results indicate that the ELISA technique we developed to measure plasmatic NE is a powerful tool for studying the role of elastase in equine inflammatory disease. In future, the application will be extended to other equine biological fluids.
Copyright 2009 Elsevier B.V. All rights reserved.
Publication Date: 2009-10-29 PubMed ID: 19932512DOI: 10.1016/j.vetimm.2009.10.023Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Validation Study
Summary
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This research study explores the development of an assay for measuring equine neutrophil elastase in horse plasma, a protein critical to inflammation and tissue damage, with a specific application in colic cases.
Study Objectives and Methodology
- The objective of this study was to create an enzyme-linked immunosorbent assay (ELISA) that can measure equine neutrophil elastase (NE) in horse plasma. This enzyme is released during inflammation and is involved in tissue destruction.
- The researchers purified elastase from equine neutrophils using double-step chromatography, resulting in a pure 27kDa protein, hypothesized to be NE 2B.
- The developed ELISA makes use of two specific polyclonal antibodies from rabbit and guinea pig to form a sandwich complex, which is detected using a secondary antibody conjugated to alkaline phosphatase.
Findings
- The developed ELISA proved to be accurate and precise, with intra- and inter-assay coefficients of variation below 10% for equine NE concentrations ranging from 1.875 to 60 ng/ml.
- NE levels in plasma remained stable despite centrifugation delays of over 4 hours when using EDTA anticoagulated blood.
- In a study involving 37 healthy horses, the average NE level was 32.53+/-4.6 ng/ml.
- NE levels in horses with colic upon admission were significantly higher than those in healthy horses, indicating the potential diagnostic value of this ELISA for colic.
Conclusion and Future Scope
- This research concludes that the developed ELISA is a powerful tool for studying the role of elastase in equine inflammatory disease, particularly with respect to conditions such as colic.
- The researchers plan on extending the application of this assay to other biological fluids of horses in the future. This extension could broaden our understanding of how NE behaves across different bodily fluids, potentially uncovering new data about equine health and disease.
Cite This Article
APA
de la Rebière de Pouyade G, Franck T, Salciccia A, Deby-Dupont G, Grulke S, Heyden LV, Sandersen C, Serteyn D.
(2009).
Development of an enzyme-linked immunosorbent assay for equine neutrophil elastase measurement in blood: preliminary application to colic cases.
Vet Immunol Immunopathol, 135(3-4), 282-288.
https://doi.org/10.1016/j.vetimm.2009.10.023 Publication
Researcher Affiliations
- Department of Clinical Sciences, Equine Clinic, Faculty of Veterinary Medicine B41, University of Liège, Sart Tilman, 4000 Liège, Belgium. G.delaRebieredePouyade@ulg.ac.be
MeSH Terms
- Animals
- Antibodies
- Colic / blood
- Colic / enzymology
- Colic / veterinary
- Enzyme-Linked Immunosorbent Assay / methods
- Enzyme-Linked Immunosorbent Assay / statistics & numerical data
- Enzyme-Linked Immunosorbent Assay / veterinary
- Guinea Pigs
- Horse Diseases / blood
- Horse Diseases / enzymology
- Horses / blood
- Leukocyte Elastase / blood
- Leukocyte Elastase / immunology
- Rabbits
- Reference Values
- Reproducibility of Results
- Sensitivity and Specificity
Citations
This article has been cited 2 times.- Sandersen C, Bienzle D, Cerri S, Franck T, Derochette S, Neven P, Mouytis-Mickalad A, Serteyn D. Effect of inhaled hydrosoluble curcumin on inflammatory markers in broncho-alveolar lavage fluid of horses with LPS-induced lung neutrophilia. Multidiscip Respir Med 2015;10(1):16.
- Derochette S, Mouithys-Mickalad A, Franck T, Collienne S, Ceusters J, Deby-Dupont G, Neven P, Serteyn D. NDS27 combines the effect of curcumin lysinate and hydroxypropyl-β-cyclodextrin to inhibit equine PKCδ and NADPH oxidase involved in the oxidative burst of neutrophils. FEBS Open Bio 2014;4:1021-9.
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