Production of monoclonal antibodies directed against the microsporidium Enterocytozoon bieneusi

Several hybridomas producing antibodies detected by indirect immunofluoresc ence antibody test (IFAT) were established by fusion of mouse myeloma SP2/O with spleen cells from BALB/c mice immunized against whole spores (protoco l 1) or chitinase-treated spores (protocol 2) of Enterocytozoon bieneusi an d were cloned twice by limiting dilutions. Two monoclonal antibodies (MAbs) , 3B82H2 from protocol 1, isotyped as immunoglobulin hi (IgM), and 6E52D9 f rom protocol 2, isotyped as IgG, were expanded in both ascites and culture. IFAT with the MAbs showed that both MAbs reacted exclusively with the wall s of the spores of E. bieneusi, strongly staining the surface of mature spo res, and produced titers of greater than 4,096. Immunogold electron microsc opy confirmed the specific reactivities of both antibodies, No cross-reacti on, either with the spores of the other intestinal microsporidium species E ncephalitozoon intestinalis or with yeast cells, bacteria, or any other int estinal parasites, was observed. The MAbs were used to identify E, bieneusi spores in fecal specimens from patients suspected of having intestinal mic rosporidiosis. The IFAT was validated against standard staining methods (Ch romotrope 2R and Uvitex 2B) and PCR, We report here the first description a nd characterization of two MAbs specific for the spore wall of E, bieneusi, These MAbs have great potential for the demonstration and species determin ation of E, bieneusi, and their application in immunofluorescence identific ation off. bieneusi in stool samples could offer a new diagnostic tool for clinical Laboratories.