Detection of microsporidia by indirect immunofluorescence antibody test using polyclonal and monoclonal

During a screening for monoclonal antibodies (MAbs) to the microsporidian Encephalitozoon hellem, three murine hybridoma cell lines producing strong enzyme-linked immunosorbent assay (ELISA) reactivities were cloned twice, were designated C12, E9, and Ell, and were found to secrete MAbs of the immunoglobulin M isotype. On subsequent ELISAs, the three MAbs reacted most strongly to E. hellem, and they reacted somewhat less to Encephalitozoon cuniculi and least to Nosema corneum, two other microsporidian species. The MAbs produced values of absorbance against microsporidia that were at least three times greater than reactivities obtained with control hybridoma supernatants or with uninfected host cell proteins used as antigens. By Western blot immunodetection, the three MAbs detected three E. helkm antigens with relative molecular weights (Mrs) of 62, 60, and 52 when assayed at the highest supernatant dilutions producing reactivity. At lower dilutions, the MAbs detected additional proteins with Mrs of 55 and 53. By using indirect immunofluorescence antibody staining, the MAbs, as well as hyperimmune polyclonal murine antisera raised against E. cuniculi and E. helem, were able to detect formalin-fixed, tissue culture-derived E. cuniculi and E. heUlem and two other human microsporidia, Enterocytozoon bieneusi and Septata intestinalis, in formalin-fixed stool and urine, respectively. E. bieneusi, however, stained more intensely with the polyclonal antisera than with the MAbs. Neither the MAbs nor the hyperimmune murine polyclonal antibodies detected Cryptosporidium, Giardia