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Journal of Clinical Microbiology, May 2001, p. 1947-1951, Vol. 39, No. 5
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.5.1947-1951.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

One-Step Purification of Enterocytozoon bieneusi Spores from Human Stools by Immunoaffinity Expanded-Bed Adsorption

Isabelle Accoceberry,1,* Marc Thellier,2 Annick Datry,2 Isabelle Desportes-Livage,2 Sylvestre Biligui,2 Martin Danis,2 and Xavier Santarelli3

Laboratoire de Parasitologie-Mycologie, Centre Hospitalier-Universitaire de Bordeaux, Hôpital Saint André, 33075 Bordeaux Cedex,1 Unité INSERM 511 and Laboratoire de Parasitologie-Mycologie, Centre Hospitalier-Universitaire de la Pitié-Salpêtrière, 75013 Paris,2 and Ecole Supérieure de Technologie des Biomolécules de Bordeaux (ESTBB) UMR 5544, Université Victor Segalen de Bordeaux 2, 33076 Bordeaux Cedex,3 France

Received 12 October 2000/Returned for modification 14 January 2001/Accepted 26 February 2001

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 107 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.

* Corresponding author. Mailing address: Laboratoire de Parasitologie-Mycologie, CHU de Bordeaux, 33000 Bordeaux, France. Phone: 33-5-56-79-58-37. Fax: 33-5-56-79-58-79. E-mail: isabelle.accoceberry{at}chu-bordeaux.fr.

Journal of Clinical Microbiology, May 2001, p. 1947-1951, Vol. 39, No. 5
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.5.1947-1951.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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