The development of a sensitive and specific ELISA for mouse eosinophil peroxidase: assessment of eosinophil degranulation ex vivo and in models of human disease.
J Immunol Methods
; 375(1-2): 138-47, 2012 Jan 31.
Article
em En
| MEDLINE
| ID: mdl-22019643
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ensaio de Imunoadsorção Enzimática
/
Degranulação Celular
/
Peroxidase de Eosinófilo
/
Proteínas Granulares de Eosinófilos
/
Eosinófilos
/
Anticorpos Monoclonais
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
J Immunol Methods
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
Estados Unidos