One-step purification of Enterocytozoon bieneusi spores from human stools by immunoaffinity expanded-bed adsorption

Abstract

An original, reliable, and reproducible method for the purification of Enterocytozoon bieneusi spores from human stools is described. We recently reported the production of a species-specific monoclonal antibody (MAb) 6E52D9 immunoglobulin G2a (IgG2a) raised against the exospore of E. bieneusi spore walls. The MAb was used as a ligand to develop an immunoaffinity matrix. The mouse IgG2a MAb was bound directly to a Streamline rProtein A adsorbent, used for expanded-bed adsorption of immunoglobulins, for optimal spatial orientation of the antibody and maximum binding efficiency of the antigen. The complex was then cross-linked covalently using dimethyl pimelimidate dihydrochloride. After incubation of the immunoaffinity matrix with filtered stool samples containing numerous E. bieneusi spores and before elution with 6 M guanidine HCl, the expansion of the adsorbent bed eliminated all the fecal contaminants. The presence of spores in the elution fractions was determined by an indirect immunofluorescence antibody test (IFAT). E. bieneusi spores were found in the elution fraction in all four experiments and were still highly antigenic as indicated by IFAT. Smears examined by light microscopy contained very clean spores with no fecal debris or background bacterial and fungal contaminants. However, spore recovery rates were relatively low: an average of 10(7) spores were purified per run. This technique for isolating E. bieneusi spores directly from human stool samples with a high degree of purity opens up new approaches for studying this parasite.

FIG. 1

Chromatogram of spore purification using the immuno-Streamline rProtein A matrix. Features: column, Streamline 25 (75 ml of gel); sample, 75 ml of the spore suspension; buffer, PBS at pH 7.2; detection at 280 nm (y axis); elution volume (x axis); flow rate, 2 ml/min in equilibration and injection (I), 32 ml/min in expansion (Ex), and 15 ml/min in sedimented bed for elution (El). The fractions between 500 and 1,500 ml of elution volume represent fecal debris eliminated after expansion. The peak at ca. 1,600 ml of elution volume represents the eluted spores.

FIG. 2

Purified whole spores of E. bieneusi in the concentrated eluted fraction of sample 2, stained by indirect immunofluorescence with MAbs 6E52D9 (A) and 3B82H2 (B). Both MAbs recognize antigens localized in the spore walls. Smears were shown to contain very clean spores with no fecal debris or background bacterial and fungal contaminants (×1,000 magnification).