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

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 off. 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 ri adsorbent, used fur expanded-bed adsorption of immonoglobulins, 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 vr ere found in the elution fraction in all four experiments and were still highly antigenic as indicated by IFAT. Smears examined hv 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.