Production and characterization of monoclonal antibodies against Enterocytozoon bieneusi purified from rhesus macaques

Abstract

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.

FIG. 1.

Preformed iodixanol gradient centrifugation. Spores purified by Percoll and sucrose gradient centrifugation resolved into one or two bands (M or B1 and B2) by 10 to 50% continuous preformed iodixanol gradient centrifugation. The sample from the Percoll middle layer resolved into one band (M) (right tube; specific density, 1.146 to 1.156 g/ml), but the sample from the Percoll bottom layer resolved into two bands (left tube; B1's specific density was equal to that of band M and B2's specific density was 1.165 to 1.175 g/ml).

FIG. 2.

Electron micrograph showing the purity (magnification, ×5,850) and the typical structures (inset; magnification, ×42,750) of spores.

FIG. 3.

Spores detected by IFA. E. bieneusi spores purified from a macaque and a human (top row) or present in positive feces from a macaque and a human (bottom row) were stained by MAb 8E2. The same results were obtained using MAbs 7G2, 7H2, and 12G8 (data not shown).

FIG. 4.

Immunoelectron micrographs of E. bieneusi spores. Partially purified spores (from macaques) were first incubated with MAbs 8E2, 7G2, 7H2, and 12G8 or with DMEM as a control (CTRL) and then stained with gold-labeled anti-mouse IgG. Gold particles appeared on the spore walls but not on bacteria or other debris. No gold particles were observed on control spores.

FIG. 5.

Blocking ELISA. The reciprocal rates of inhibition demonstrate that the epitopes recognized by MAbs 8E2, 7G2/7H2, and 12G8 are different.

FIG. 6.

Western blot analysis of reduced E. bieneusi spore proteins. Lane 1, molecular weight marker; lane 2, mouse preimmune serum; lane 3, mouse immune serum; lane 4, medium as a blank; lane 5, 8E2; lane 6, 7G2; lane 7, 7H2; and lane 8, 12G8.