Development of an enzyme-linked immunosorbent assay for specific equine neutrophil myeloperoxidase measurement in blood.
Abstract: Equine inflammatory disease is accompanied by a neutrophil activation resulting in the release of granulocytic enzyme myeloperoxidase (MPO). To measure MPO in horse plasma as marker of neutrophil activation, the authors purified equine neutrophil MPO and developed a specific enzyme immunoassay using 2 specific polyclonal antibodies obtained from rabbit (primary antibody) and guinea pig (secondary antibody). The sandwich complex "primary antibody-MPO-secondary antibody" was detected using a goat anti-guinea pig immunoglobulin antibody conjugated to alkaline phosphatase. The enzyme-linked immunosorbent assay (ELISA) showed good precision and accuracy, with intra- and interassay coefficients of variation below 10% for MPO concentrations ranging from 0.78 to 50 ng/ml. A stable plasma MPO value, unaffected by time elapsed between blood collection and centrifugation, was obtained with plasma from EDTA anticoagulated blood. The mean MPO value measured in 38 healthy horses was 181.80 +/- 64.74 ng/ml. In 20 horses suffering from obstruction of the large or small intestine, MPO concentrations measured at the time of arrival at the intensive care unit were significantly higher than mean normal value, ranging from 477.88 to 2,748.13 ng/ml. Work is in progress to apply this MPO ELISA technique to other biological fluids and other equine diseases.
Publication Date: 2005-11-30 PubMed ID: 16312231DOI: 10.1177/104063870501700502Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Antibodies
- Blood
- Clinical Study
- Diagnosis
- Diagnostic Technique
- Disease Diagnosis
- Disease Etiology
- Enzyme-Linked Immunosorbent Assay (ELISA)
- Enzymes
- Equine Diseases
- Equine Health
- Immune Response
- Immune System
- In Vivo
- Inflammation
- Laboratory Methods
- Neutrophils
- Plasma
- Veterinary Care
- Veterinary Medicine
- Veterinary Research
Summary
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The research involves the development of an accurate and precise test (ELISA) for assessing the release of myeloperoxidase (MPO), an enzyme released during inflammation, in horse plasma. This test was successfully used to distinguish healthy horses from those suffering from intestinal obstruction, indicating its potential in diagnosing and monitoring disease in horses.
Development of ELISA for Equine Myeloperoxidase Measurement
- The study aimed to create a reliable method for measuring the levels of enzyme called myeloperoxidase (MPO) in horse plasma. MPO is generally released as a result of neutrophil activation, which occurs during inflammation in equine diseases.
- To achieve this, the scientists developed a specific enzyme immunoassay. Basically, an immunoassay is a biochemical test that measures the presence or concentration of a substance in a solution using the concept of antibodies. In this case, they used two specific polyclonal antibodies sourced from rabbit and guinea pig. These antibodies bind specifically to MPO in what is called a “sandwich complex” – the primary rabbit antibody binds to the MPO first, and then the secondary guinea pig antibody binds to the first rabbit antibody.
- The sandwich complex consisting of the primary antibody (from rabbit), MPO and secondary antibody (from guinea pig) was then detected using another antibody derived from a goat. This goat antibody was attached to an alkaline phosphatase, making it possible to detect the presence and concentration of MPO.
Accuracy and Precision of the ELISA
- The newly developed enzyme-linked immunosorbent assay (ELISA) demonstrated good accuracy and reliability. The intra- and interassay variance rates were under 10% for MPO concentrations between 0.78 and 50 ng/ml – this means that the assay was consistent and reproducible within and across separate runs, thus indicating its reliability.
- Moreover, the researchers found that the plasma MPO value was stable and unaffected by the duration between when blood was collected and when it was centrifuged, when the blood was anticoagulated with EDTA. This stability further adds to the test’s reliability.
Application of the ELISA to Equine Health
- Testing the ELISA on 38 healthy horses, the researchers found the mean MPO value to be 181.80 ± 64.74 ng/ml.
- However, in horses suffering from either large or small intestinal obstruction, MPO levels measured upon their arrival at the intensive care unit were significantly higher than this mean normal value, ranging from 477.88 to 2,748.13 ng/ml.
- This suggests the potential of this developed MPO ELISA technique as a diagnostic or monitoring tool for equine diseases that involve inflammation and neutrophil activation.
- Future investigations will see this MPO ELISA technique applied to other biological fluids and other equine diseases.
Cite This Article
APA
Franck T, Grulke S, Deby-Dupont G, Deby C, Duvivier H, Peters F, Serteyn D.
(2005).
Development of an enzyme-linked immunosorbent assay for specific equine neutrophil myeloperoxidase measurement in blood.
J Vet Diagn Invest, 17(5), 412-419.
https://doi.org/10.1177/104063870501700502 Publication
Researcher Affiliations
- Department of General Anesthesia and Surgical Pathology of Large Animals, Institut Vétérinaire, Université de Liège, Liège, Belgium.
MeSH Terms
- Animals
- Enzyme-Linked Immunosorbent Assay / methods
- Enzyme-Linked Immunosorbent Assay / veterinary
- Horse Diseases / blood
- Horse Diseases / enzymology
- Horses
- Intestinal Obstruction / blood
- Intestinal Obstruction / enzymology
- Intestinal Obstruction / veterinary
- Neutrophils / enzymology
- Peroxidase / blood
Citations
This article has been cited 15 times.- Parrilla Hernández S, Franck T, Munaut C, Feyereisen É, Piret J, Farnir F, Reigner F, Barrière P, Deleuze S. Characterization of Myeloperoxidase in the Healthy Equine Endometrium. Animals (Basel) 2023 Jan 21;13(3).
- Franck T, Ceusters J, Graide H, Niesten A, Duysens J, Mickalad AM, Serteyn D. Equine Muscle Derived Mesenchymal Stem Cells Loaded with Water-Soluble Curcumin: Modulation of Neutrophil Activation and Enhanced Protection against Intracellular Oxidative Attack. Int J Mol Sci 2023 Jan 5;24(2).
- Janssen P, Tosi I, Hego A, Maréchal P, Marichal T, Radermecker C. Neutrophil Extracellular Traps Are Found in Bronchoalveolar Lavage Fluids of Horses With Severe Asthma and Correlate With Asthma Severity. Front Immunol 2022;13:921077.
- Storms N, Medina Torres C, Franck T, Sole Guitart A, de la Rebière G, Serteyn D. Presence of Myeloperoxidase in Lamellar Tissue of Horses Induced by an Euglycemic Hyperinsulinemic Clamp. Front Vet Sci 2022;9:846835.
- Franck T, Ceusters J, Graide H, Mouithys-Mickalad A, Serteyn D. Muscle Derived Mesenchymal Stem Cells Inhibit the Activity of the Free and the Neutrophil Extracellular Trap (NET)-Bond Myeloperoxidase. Cells 2021 Dec 10;10(12).
- Mouithys-Mickalad A, Storms N, Franck T, Ceusters J, de la Rebière de Pouyade G, Deby-Dupont G, Serteyn D. Effects of Juglone on Neutrophil Degranulation and Myeloperoxidase Activity Related to Equine Laminitis. Front Vet Sci 2021;8:677675.
- Wen Y, Yuan J, Chen J, Zhao Y, Niu Y, Yu C. Amperometric myeloperoxidase immunoassay based on the use of CuPdPt nanowire networks. Mikrochim Acta 2017 Dec 18;185(1):55.
- Boly R, Franck T, Kohnen S, Lompo M, Guissou IP, Dubois J, Serteyn D, Mouithys-Mickalad A. Evaluation of Antiradical and Anti-Inflammatory Activities of Ethyl Acetate and Butanolic Subfractions of Agelanthus dodoneifolius (DC.) Polhill & Wiens (Loranthaceae) Using Equine Myeloperoxidase and Both PMA-Activated Neutrophils and HL-60 Cells. Evid Based Complement Alternat Med 2015;2015:707524.
- Li X, Zhou X, Ye Y, Li Y, Li J, Privratsky B, Wu E, Gao H, Huang C, Wu M. Lyn regulates inflammatory responses in Klebsiella pneumoniae infection via the p38/NF-κB pathway. Eur J Immunol 2014 Mar;44(3):763-73.
- Tsumbu CN, Deby-Dupont G, Tits M, Angenot L, Frederich M, Kohnen S, Mouithys-Mickalad A, Serteyn D, Franck T. Polyphenol content and modulatory activities of some tropical dietary plant extracts on the oxidant activities of neutrophils and myeloperoxidase. Int J Mol Sci 2012;13(1):628-650.
- Verwilghen DR, Martens A, Busschers E, Franck T, Deberg M, Henrotin Y, Vanderheyden L, Serteyn D. Coll2-1, Coll2-1NO2 and myeloperoxidase concentrations in the synovial fluid of equine tarsocrural joints affected with osteochondrosis. Vet Res Commun 2011 Oct;35(7):401-8.
- Grulke S, Franck T, Gangl M, Péters F, Salciccia A, Deby-Dupont G, Serteyn D. Myeloperoxidase assay in plasma and peritoneal fluid of horses with gastrointestinal disease. Can J Vet Res 2008 Jan;72(1):37-42.
- Art T, Franck T, Lekeux P, de Moffarts B, Couëtil L, Becker M, Kohnen S, Deby-Dupont G, Serteyn D. Myeloperoxidase concentration in bronchoalveolar lavage fluid from healthy horses and those with recurrent airway obstruction. Can J Vet Res 2006 Oct;70(4):291-6.
- Mydel P, Takahashi Y, Yumoto H, Sztukowska M, Kubica M, Gibson FC 3rd, Kurtz DM Jr, Travis J, Collins LV, Nguyen KA, Genco CA, Potempa J. Roles of the host oxidative immune response and bacterial antioxidant rubrerythrin during Porphyromonas gingivalis infection. PLoS Pathog 2006 Jul;2(7):e76.
- Degotte G, Frederich M, Francotte P, Franck T, Colson T, Serteyn D, Mouithys-Mickalad A. Targeting Myeloperoxidase Activity and Neutrophil ROS Production to Modulate Redox Process: Effect of Ellagic Acid and Analogues. Molecules 2023 Jun 2;28(11).
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