UBC Theses and Dissertations
Cryptosporidium detection through antibody immobilization on a solid surface Das, Rony
Current detection of pathogenic organisms in water relies on the use of indicators (turbidity, Escherichia coli, total coliforms, fecal coliforms etc.) or time consuming assays that can only be done in a specialized laboratory. In this research a simple assay was developed for rapid and sensitive recovery and identification of waterborne pathogens from environmental samples. The assay developed involves capturing target pathogens onto an activated capture surface, exposing the capture surface to antibody conjugated micro-retroreflectors, and then inserting the capture surface into an inexpensive, simple reader to detect the retroreflection signal to confirm the presence of target pathogens. Antibody (capture molecule: IgG and IgM) fragments specific to Cryptosporidium, as a model waterborne pathogen, were produced and immobilized site specifically and randomly onto gold-coated surfaces as well as corner cube micro-retroreflectors (ccμRR). A shear test performed to determine the critical shear stress that antigen-antibody bonds are able to endure showed that the organism-antibody bond could resist up to a shear stress of 126 dyne/cm2 and beyond this critical value immobilized Cryptosporidium oocysts were lost from the system. Capture tests were designed to determine the optimum operating conditions of the parallel flow sampling device using IgG-Fab', IgG-Fab, and IgM-Fab' activated surfaces. Capture efficiencies did not differ significantly within the range of flow rate used (14 – 42 mL/min), but improvement was noticed when the cell depth was decreased from 250 μm to 125 μm. Site specifically oriented IgG-Fab' activated surfaces resulted in significantly better capture efficiencies than surface with randomly immobilized IgG-Fab fragments. The capture efficiency of IgG-Fab' activated surfaces was significantly different than that of IgM-Fab' immobilized surfaces. There was no significant difference in capture efficiency using the surfaces activated with dilutions (1:8, 1:4, 1:2, and 1:1.5) of antibody fragments originally made of 500μg of IgG1 antibody. The use of BSA as a blocking agent improved the reproducibility of the capture efficiency. CcμRR were suspended in solution and activated with IgG-Fab' to use as a recognition molecule. The current set up of the detector system is capable of detecting the presence and absence of a large number of ccμRR attached on the solid surface.
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