B C Ferrari1, G Vesey, K A Davis, M Gauci, D Veal. 1. Centre for Fluorimetric Applications in Biotechnology, Department of Biological Sciences, Macquarie University, Sydney, Australia.
Abstract
BACKGROUND: Cryptosporidium is an important waterborne pathogen. Detection of Cryptosporidium in concentrated water samples depends on oocyst isolation using immunomagnetic separation (IMS) and/or fluorescence-activated cell sorting (FACS), followed by confirmation using immunofluorescence staining (IFA) and fluorescence microscopy. These methods require highly trained microscopists for oocyst identification and confirmation. Analysis is hampered due to the presence of autofluorescent particles coupled with particles binding nonspecifically with the monoclonal antibodies (mAbs) used for detection. Flow cytometry (FCM) has the potential to be a more specific method for oocyst detection, but such a system would require more than one selection parameter. METHODS: Various mAbs from commercial suppliers were paired with CRY104-PE and evaluated. The mAb combination that best discriminated stained oocyst from detritus was optimized and compared to Cryptosporidium detection utilizing one-color IFA/FACS. RESULTS: A highly specific two-color assay employing the IgG(1) mAb CRY104 was developed. The assay resulted in reductions, up to 20-fold, in the number of non-Cryptosporidium particles detected. The addition of a second selection parameter improved microscopic analysis times and simplified oocyst confirmation by microscopists. CONCLUSIONS: A two-color assay employing competing surface mAbs reduces the number of fluorescent particles sorted, thus improving FCM detection methods for Cryptosporidium. Copyright 2000 Wiley-Liss, Inc.
BACKGROUND: Cryptosporidium is an important waterborne pathogen. Detection of Cryptosporidium in concentrated water samples depends on oocyst isolation using immunomagnetic separation (IMS) and/or fluorescence-activated cell sorting (FACS), followed by confirmation using immunofluorescence staining (IFA) and fluorescence microscopy. These methods require highly trained microscopists for oocyst identification and confirmation. Analysis is hampered due to the presence of autofluorescent particles coupled with particles binding nonspecifically with the monoclonal antibodies (mAbs) used for detection. Flow cytometry (FCM) has the potential to be a more specific method for oocyst detection, but such a system would require more than one selection parameter. METHODS: Various mAbs from commercial suppliers were paired with CRY104-PE and evaluated. The mAb combination that best discriminated stained oocyst from detritus was optimized and compared to Cryptosporidium detection utilizing one-color IFA/FACS. RESULTS: A highly specific two-color assay employing the IgG(1) mAb CRY104 was developed. The assay resulted in reductions, up to 20-fold, in the number of non-Cryptosporidium particles detected. The addition of a second selection parameter improved microscopic analysis times and simplified oocyst confirmation by microscopists. CONCLUSIONS: A two-color assay employing competing surface mAbs reduces the number of fluorescent particles sorted, thus improving FCM detection methods for Cryptosporidium. Copyright 2000 Wiley-Liss, Inc.