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Infection and Immunity, August 2005, p. 5166-5172, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.5166-5172.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Production and Characterization of Monoclonal Antibodies against Enterocytozoon bieneusi Purified from Rhesus Macaques

Division of Infectious Diseases, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536,¹ New England Regional Primate Research Center, Southborough, Massachusetts 01772²

Received 11 February 2005/ Returned for modification 28 February 2005/ Accepted 18 March 2005

Enterocytozoon bieneusi spores derived from rhesus macaque feces were purified by serial salt-Percoll-sucrose-iodixanol centrifugation, resulting in two bands with different specific densities of 95.6% and 99.5% purity and with a recovery efficiency of 10.8%. An ultrastructural examination revealed typical E. bieneusi spores. Twenty-six stable hybridomas were derived from BALB/c mice immunized with spores and were cloned twice by limiting dilution or growth on semisolid medium. Four monoclonal antibodies (MAbs), reacting exclusively with spores, were further characterized. These MAbs specifically reacted with spores present in stools of humans and macaques, as visualized by immunofluorescence, and with spore walls, as visualized by immunoelectron microscopy. A blocking enzyme-linked immunosorbent assay and Western blotting revealed that the epitope recognized by 8E2 was different from those recognized by 7G2, 7H2, and 12G8, which identified the same 40-kDa protein. These MAbs will be valuable tools for diagnostics, for epidemiological investigations, for host-pathogen interaction studies, and for comparative genomics and proteomics.

Editor: W. A. Petri, Jr.

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