Toxoplasma gondii major surface antigen (SAG1): in vitro analysis of host cell binding

Previous studies have indicated that SAG1, the major surface molecule of the protozoan parasite Toxoplasma gondii, is an important attachment ligand for the host cell. However, the research data that supports this claim comes largely from studies investigating tachyzoite binding, and not SAG1 binding per se. In this study we successfully developed an in vitro attachment assay to directly evaluate the mechanism of SAG1-host cell binding. Competition experiments were then performed using SAG1 that had been pre-treated with the neoglycoprotein BSA-glucosamide or with antibody. Soluble BSA-glucosamide blocked SAG1 attachment to MDBK cells in a dose-dependent manner, implying that SAG1 binding is mediated, in part, via attachment to host cell surface glucosamine. Interestingly, pre-incubation of SAG1 in polyclonal sera from chronically infected mice failed to block binding. This challenges the assumption that anti-SAG1 antibodies block parasite attachment through the masking of SAG1 host cell binding domains. Taken together, this evidence presents new strategies for understanding SAG1-mediated attachment

The development and optimisation of Nanobody based electrochemical immunosensors for IgG

Biosensors are increasingly heralded for their potential to create inexpensive diagnostic devices which are sensitive, selective and easy to use. One of the key categories of biosensor are immunosensors, which have historically used antibodies as bioreceptors. Though widely used, antibodies bring inherent limitations such as variability, limited stability and their reliance on animal sources. This has led to the development of alternative immuno-reagents such as non-antibody binding proteins (NABPs). These are low molecular weight proteins which largely avoid the aforementioned advantages of antibodies. They are commonly produced by bacteria enabling the use of DNA technology to manipulate bioreceptors at the molecular level. Single chain VHHs (commonly known as nanobodies) are an antibody derived NABP adapted from camelid heavy chain antibodies which are the isolated binding domain. Whilst nanobodies have been used for diagnostic and therapeutic applications, they have limited demonstration in biosensors. In this study, both antibodies and nanobodies were used to construct a biosensor. In addition nanobody performance was optimised by introducing a novel peptide spacer. The role of nanobody orientation and spacing was thus investigated and spacer length was optimised, leading to an increase in the sensitivity of the biosensor