A sensitive high throughput ELISA for human eosinophil peroxidase: A specific assay to quantify eosinophil degranulation from patient-derived sources

Sergei I. Ochkur, John Dongil Kim, Cheryl A. Protheroe, Dana Colbert, Rachel M. Condjella, Sophie Bersoux, Richard A. Helmers, Redwan Moqbel, Paige Lacy, Elizabeth A. Kelly, Nizar N. Jarjour, Robert Kern, Anju Peters, Robert P. Schleimer, Glenn T. Furuta, Parameswaran Nair, James J. Lee, Nancy A. Lee

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patients in a clinical setting have been limited to ELISA assessments for the presence of the prominent granule ribonucleases, ECP and EDN. However, the demonstration that these ribonucleases are expressed by leukocytes other than eosinophils, as well as cells of non-hematopoietic origin, limits the usefulness of these assays. Two novel monoclonal antibodies recognizing eosinophil peroxidase (EPX) were used to develop an eosinophil-specific and sensitive sandwich ELISA. The sensitivity of this EPX-based ELISA was shown to be similar to that of the commercially available ELISA kits for ECP and EDN. More importantly, evidence is also presented confirming that among these granule protein detection options, EPX-based ELISA is the only eosinophil-specific assay. The utility of this high throughput assay to detect released EPX was shown in ex vivo degranulation studies with isolated human eosinophils. In addition, EPX-based ELISA was used to detect and quantify eosinophil degranulation in several in vivo patient settings, including bronchoalveolar lavage fluid obtained following segmental allergen challenge of subjects with allergic asthma, induced sputum derived from respiratory subjects following hypotonic saline inhalation, and nasal lavage of chronic rhinosinusitis patients. This unique EPX-based ELISA thus provides an eosinophil-specific assay that is sensitive, reproducible, and quantitative. In addition, this assay is adaptable to high throughput formats (e.g., automated assays utilizing microtiter plates) using the diverse patient fluid samples typically available in research and clinical settings.

Original languageEnglish (US)
Pages (from-to)10-20
Number of pages11
JournalJournal of Immunological Methods
Volume384
Issue number1-2
DOIs
StatePublished - Oct 31 2012

Keywords

  • Allergic inflammation
  • EPX
  • Eosinophilia
  • Granule proteins

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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    Ochkur, S. I., Kim, J. D., Protheroe, C. A., Colbert, D., Condjella, R. M., Bersoux, S., Helmers, R. A., Moqbel, R., Lacy, P., Kelly, E. A., Jarjour, N. N., Kern, R., Peters, A., Schleimer, R. P., Furuta, G. T., Nair, P., Lee, J. J., & Lee, N. A. (2012). A sensitive high throughput ELISA for human eosinophil peroxidase: A specific assay to quantify eosinophil degranulation from patient-derived sources. Journal of Immunological Methods, 384(1-2), 10-20. https://doi.org/10.1016/j.jim.2012.06.011