Isolation of Affimers against biomarkers of Clostridium difficile infection for use as diagnostic tools

Clostridium difficile is one of the leading causal agents of hospital acquired infection and antibiotic-associated diarrhoea. The treatment and control of Clostridium difficile infection (CDI) is critically dependent on accurate laboratory diagnosis. However, current diagnostic methods have limitations including cost, potential over-sensitivity, lack of detection of toxin protein associated with nucleic acid amplification techniques, and long turnaround time for toxigenic cultures. Detection of toxins in faecal samples of patients suffering from CDI is a highly significant and necessary criterion for the diagnosis of CDI. Rapid enzyme immunoassays are used for toxin detection and can be completed in less than an hour, however, their low sensitivities make them unacceptable for use as a stand-alone test. To date, no one-step diagnostic that is low cost, sensitive and specific is available for CDI diagnosis. Leeds has developed a non-antibody binding protein called Affimer type II (Affimer). From phage display libraries, Affimer binders against >350 targets have been identified.
This thesis investigates the isolation of Affimers against biomarkers of Clostridium difficile infection for use as diagnostic tools. Phage display screening yielded high affinity Affimers against the three well-established biomarkers of CDI (toxin A, toxin B and glutamate dehydrogenase). Characterisation of the Affimer binders show that they bind to their target with low nanomolar affinity. Through sandwich phage ELISA, two toxin B Affimers have been established for use as a pair in sandwich assay format. This thesis has also explored the ability of Affimers to function as novel reagents for the potential development of a point-of-care diagnostic tool for C. difficile infection. The most exciting result include the development of a toxin B hybrid assay which shows improved sensitivity and specificity by switching one of the molecular recognition elements of a clinically used C. difficile detection kit from antibodies to Affimers.