Detection of mouse eosinophils and quantitative assessments of degranulation using secondary granule protein-specific polyclonal and monoclonal antibodies

Abstract

Rationale Tissue eosinophilia is an accepted hallmark feature of parasite infections and allergen-mediated inflammatory responses. We have developed a number of polyclonal and monoclonal antibodies specific for mouse eosinophils and their associated products to facilitate studies of this leukocyte and the use of mouse models of human diseases. Methods Eosinophil secondary granule proteins, including major basic protein (MBP), eosinophil peroxidase (EPO), and eosinophil associated ribonucleases (EARs), were purified from eosinophils derived from IL-5 transgenic mice. These purified proteins were used in immunization and screening protocols to generate rabbit polyclonal antisera as well as monoclonal antibodies in rats and mice. Results Rabbit polyclonal antisera reactive to MBP and EARs were generated and shown to be useful reagents in western blots of cell/tissue extracts, immuno-electron microscopy, and immunohistochemistry/immunofluorescence using formalin-fixed paraffin-embedded tissue sections. Moreover, a series of rat and mouse monoclonal antibodies reactive to MBP, EPO, and EARs were produced and shown also to function in the venues noted above. Many of these antibodies also cross-react with human and other non-rodent eosinophil targets. Finally, specific monoclonal antibodies were selected for their use in an immunoblot ELISA format which has yielded a quantitative assay to measure the levels of released granule components in compartments such as the airway lumen (i.e., BAL). Conclusions The availability of multiple eosinophil-specific antibody reagents provides the community with invaluable resources for the characterization of eosinophils and their role(s) in disease model systems. Information regarding specific reagents, as well as their availability, is possible through a newly established website http://www.eosinophils.org.