TY - JOUR
T1 - A sensitive high throughput ELISA for human eosinophil peroxidase
T2 - A specific assay to quantify eosinophil degranulation from patient-derived sources
AU - Ochkur, Sergei I.
AU - Kim, John Dongil
AU - Protheroe, Cheryl A.
AU - Colbert, Dana
AU - Condjella, Rachel M.
AU - Bersoux, Sophie
AU - Helmers, Richard A.
AU - Moqbel, Redwan
AU - Lacy, Paige
AU - Kelly, Elizabeth A.
AU - Jarjour, Nizar N.
AU - Kern, Robert
AU - Peters, Anju
AU - Schleimer, Robert P.
AU - Furuta, Glenn T.
AU - Nair, Parameswaran
AU - Lee, James J.
AU - Lee, Nancy A.
N1 - Funding Information:
The authors wish to thank the members of all the participating laboratories as well as colleagues within the greater eosinophil community for insightful discussions and critical comments that directly led to the development of an EPX-based ELISA with clinically utility. We also wish to acknowledge the invaluable assistance of the Mayo Clinic Arizona Statistical support group (Amylou Dueck, PhD, Yu-Hui J. Chang, and Joseph Hentz), our staff medical graphic artist (Marv Ruona), the Mayo Clinic Arizona Phlebotomy System Staff (Marian Gannon and Graciela Snyder), the members of the Northwestern Sinus Center, and the excellent administrative support provided to Lee Laboratories by Linda Mardel and Shirley (“Charlie”) Kern. The Mayo Foundation for Medical Education and Research and grants from the United States National Institutes of Health [NAL ( HL058723 ), JJL ( HL065228 , RR0109709 ), RPS ( HL068546 , HL078860 ), NNJ ( HL088594 , RR025011 , HL56396 , RR03186 ), GTF ( RR025780 )], the American Heart Association [NAL ( 05556392 ) and JJL ( 0855703 )], The Bazley Trust (RPS), the Canadian Institutes of Health Research [RM and PL ( MOP89748 )], the Lung Association of Alberta [JDK], and a Canada Research Chair in Airway Inflammometry (PN) were the sources of funding used in the performance of studies including data analysis and manuscript preparation. These funding sources had no involvement in study design, data collection (including analysis and interpretation), the writing of the manuscript, or the decision to submit for publication.
PY - 2012/10/31
Y1 - 2012/10/31
N2 - 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.
AB - 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.
KW - Allergic inflammation
KW - EPX
KW - Eosinophilia
KW - Granule proteins
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U2 - 10.1016/j.jim.2012.06.011
DO - 10.1016/j.jim.2012.06.011
M3 - Article
C2 - 22750539
AN - SCOPUS:84865587245
SN - 0022-1759
VL - 384
SP - 10
EP - 20
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -