title: Development of a Cryptosporidium oocyst assay using an automated fiber optic-based biosensor.
abstract: An intestinal protozoan parasite, Cryptosporidium parvum, is a major cause of waterborne gastrointestinal disease worldwide. Detection of Cryptosporidium oocysts in potable water is a high priority for the water treatment industry to reduce potential outbreaks among the consumer populace. Anti-Cryptosporidium oocyst polyclonal and monoclonal antibodies were tested as capture and detection reagents for use in a fiber optic biosensor assay for the detection of Cryptosporidium oocysts. Antibodies were validated using enzyme-linked immunosorbent assays, flow cytometry, Western blotting and fluorescent microscopy. Oocysts could be detected at a concentration of 105 oocysts/ml when the polyclonal antibodies were used as the capture and detection reagents. When oocysts were boiled prior to detection, a ten-fold increase in sensitivity was achieved using the polyclonal antibody. Western blotting and immunofluorescence revealed that both the monoclonal and polyclonal antibodies recognize a large (>300 kDa) molecular weight mucin-like antigen present on the surface of the oocyst wall. The polyclonal antibody also reacted with a small (105 kDa) molecular weight antigen that was present in boiled samples of oocysts. Preliminary steps to design an in-line biosensor assay system have shown that oocysts would have to be concentrated from water samples and heat treated to allow detection by a biosensor assay.
authors.affiliation: Division of Cell Biology, Microbiology, and Molecular Biology, Department of Biology, University of South Florida, 4202 E, Fowler Ave, SCA110, Tampa, FL 33620, USA. mariannefbrannon@gmail.com
authors.forename: Marianne F
authors.initials: MF
authors.lastname: Kramer
authors.forename: Graham
authors.initials: G
authors.lastname: Vesey
authors.forename: Natasha L
authors.initials: NL
authors.lastname: Look
authors.forename: Ben R
authors.initials: BR
authors.lastname: Herbert
authors.forename: Joyce M
authors.initials: JM
authors.lastname: Simpson-Stroot
authors.forename: Daniel V
authors.initials: DV
authors.lastname: Lim