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.

Investigation of Cryptosporidium spp. antigen by ELISA method in stool specimens obtained from patients with diarrhea

“…Twenty different species belonging to this genus have been isolated from mammals (vertebrates), birds, reptiles, and different species of fish. The best known species are Cryptosporidium parvum, Cryptosporidium baileyi, and Cryptosporidium muris (Akısü and Korkmaz 2005;Kramer et al 2007;Özcel et al 2007).…”

Recent developments in detection and enumeration of waterborne bacteria: a retrospective minireview

“…RAPTOR system developed by the US Naval Research Laboratory based on wave fluoroimmunoassay allows detection of waterborne pathogens simultaneously at concentrations of 5 × 10 4 Giardia lamblia cysts/mL (Anderson and Rowe‐Tait ), 10 4 CFU/mL E. coli O157:H7 (Lim ), 10 5 Cryptosporidium parvum oocysts/mL (Kramer et al. ), fecal indicators such as enterococci in environmental samples at 10 5 CFU/mL (Leskinen and Lim ) and 10 3 CFU/mL Salmonella enterica Enteritidis (Valadez et al. ).…”

Aptamer-Based Technologies for Parasite Detection

“…While FOBS have been used previously to detect Cryptosporidium parvum [ 144 ], Plasmodium falciparum [ 145 ], and Giardia lamblia [ 146 ], the only aptamer-based FOB has been developed against Plasmodium falciparum to detect Pf GDH [ 53 ]. This aptasensor included a gold-sputtered U-bent POF functionalized with the previously mentioned Pf GDH-specific NG3 aptamer.…”