TY - JOUR
T1 - The development of a sensitive and specific ELISA for mouse eosinophil peroxidase
T2 - Assessment of eosinophil degranulation ex vivo and in models of human disease
AU - Ochkur, Sergei I.
AU - Kim, John Dongil
AU - Protheroe, Cheryl A.
AU - Colbert, Dana
AU - Moqbel, Redwan
AU - Lacy, Paige
AU - Lee, James J.
AU - Lee, Nancy A.
N1 - Funding Information:
The authors wish to thank the members of Lee Laboratories as well as colleagues within the greater eosinophil community for insightful discussions and critical comments that directly led to the development of the EPX-based ELISA and, in turn, the preparation of this manuscript. 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), and the excellent administrative support provided to Lee Laboratories by Linda Mardel and Shirley (“Charlie”) Kern. The Mayo Foundation and grants from the United States National Institutes of Health [NAL ( HL058723 ) and JJL ( HL065228, RR0109709 )], the American Heart Association [NAL ( 05556392 ) and JJL ( 0855703 )], the Canadian Institutes of Health Research [RM ( MOP89748 )], and the Lung Association of Alberta [JDK] were the sources of funding used in the performance of studies as well as data analysis. 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 - 2012/1/31
Y1 - 2012/1/31
N2 - Mouse models of eosinophilic disorders are often part of preclinical studies investigating the underlying biological mechanisms of disease pathology. The presence of extracellular eosinophil granule proteins in affected tissues is a well established and specific marker of eosinophil activation in both patients and mouse models of human disease. Unfortunately, assessments of granule proteins in the mouse have been limited by the availability of specific antibodies and a reliance on assays of released enzymatic activities that are often neither sensitive nor eosinophil specific. The ability to detect immunologically and quantify the presence of a mouse eosinophil granule protein in biological fluids and/or tissue extracts was achieved by the generation of monoclonal antibodies specific for eosinophil peroxidase (EPX). This strategy identified unique pairs of antibodies with high avidity to the target protein and led to the development of a unique sandwich ELISA for the detection of EPX. Full factorial design was used to develop this ELISA, generating an assay that is eosinophil-specific and nearly 10 times more sensitive than traditional OPD-based detection methods of peroxidase activity. The added sensitivity afforded by this novel assay was used to detect and quantify eosinophil degranulation in several settings, including bronchoalveolar fluid from OVA sensitized/challenged mice (an animal model of asthma), serum samples derived from peripheral blood recovered from the tail vasculature, and from purified mouse eosinophils stimulated ex vivo with platelet activating factor (PAF) and PAF. +. ionomycin. This ability to assess mouse eosinophil degranulation represents a specific, sensitive, and reproducible assay that fulfills a critical need in studies of eosinophil-associated pathologies in mice.
AB - Mouse models of eosinophilic disorders are often part of preclinical studies investigating the underlying biological mechanisms of disease pathology. The presence of extracellular eosinophil granule proteins in affected tissues is a well established and specific marker of eosinophil activation in both patients and mouse models of human disease. Unfortunately, assessments of granule proteins in the mouse have been limited by the availability of specific antibodies and a reliance on assays of released enzymatic activities that are often neither sensitive nor eosinophil specific. The ability to detect immunologically and quantify the presence of a mouse eosinophil granule protein in biological fluids and/or tissue extracts was achieved by the generation of monoclonal antibodies specific for eosinophil peroxidase (EPX). This strategy identified unique pairs of antibodies with high avidity to the target protein and led to the development of a unique sandwich ELISA for the detection of EPX. Full factorial design was used to develop this ELISA, generating an assay that is eosinophil-specific and nearly 10 times more sensitive than traditional OPD-based detection methods of peroxidase activity. The added sensitivity afforded by this novel assay was used to detect and quantify eosinophil degranulation in several settings, including bronchoalveolar fluid from OVA sensitized/challenged mice (an animal model of asthma), serum samples derived from peripheral blood recovered from the tail vasculature, and from purified mouse eosinophils stimulated ex vivo with platelet activating factor (PAF) and PAF. +. ionomycin. This ability to assess mouse eosinophil degranulation represents a specific, sensitive, and reproducible assay that fulfills a critical need in studies of eosinophil-associated pathologies in mice.
KW - Allergic inflammation
KW - EPX
KW - Eosinophilia
KW - Granule proteins
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U2 - 10.1016/j.jim.2011.10.002
DO - 10.1016/j.jim.2011.10.002
M3 - Article
C2 - 22019643
AN - SCOPUS:84855344697
SN - 0022-1759
VL - 375
SP - 138
EP - 147
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -