Examination of binding elements and conditions of Cryptosporidium parvum oocysts to assess its detection potential in water

Abstract

Cryptosporidium parvum is an intestinal parasite that can be spread through environmental and recreational waters, most often in the form of oocysts. As an oocyst, this parasite is resistant to chlorine disinfection, and it is the known cause of the diarrheal disease Cryptosporidiosis. In this thesis, binding elements like antibodies and aptamers are studied and compared with the purpose of assessing their binding under different conditions and therefore their potential as biorecognition elements to detect C. parvum in water. Several methods have been used to fulfill this purpose. Flow cytometry and imaging flow cytometry was used to compare and assess the binding of commercial fluorescently labelled antibodies, and aptamers reported in literature, under different conditions such as pH, inactivation procedures or heat-inactivation contact time. Horseradish peroxidase (HRP)-labelled antibodies was used to assess the colorimetric potential for Cryptosporidium detection both as a part of an in-filter detecting scheme and to assess the binding differences between live and inactivated Cryptosporidium. It was found that antibodies had better binding affinity than aptamers for Cryptosporidium that was inactivated under different methods such as desiccation, heat-treatment, freeze-treatment, and formalin-treatment. Furthermore, aptamers show an increase of non-specific binding under low pH conditions. An in-filter detection method involving the preincubation of HRP-labelled antibodies with the target and passing this solution through a syringe filter followed by the addition of 3,3',5,5'-Tetramethylbenzidine (TMB), which could allow visual observation of a color change, was also explored. This biosensor was developed as part of a detection scheme to avoid any detection sensitivity loss due to poor recovery from filters and was meant to help make detection simpler for the public. The limit of detection of this approach was found out to be 105 oocysts/mL . While this limit of detection is rather high, improvements could be made by changing the labelling of the antibodies or by changing the type of antibodies.