Team:Queens Canada/Background

Source of Antibody Sequence

The time-consuming production and screening for scFv and Fab can be overcome by using previously described sequences. We will be using protein sequences of an anti-THC scFv, and an anti-THC Fab that are available in the public domain (1, 2). The anti-THC Fab has previously shown to be specific to THC in a competitive ELISA, where increasing concentrations of THC were used in BSA-THC coated wells Likewise, the anti-THC scFv is specific to THC Moreover, both the scFv and the Fab were designed to be expressed in E. coli.

However, neither antibody is conjugated to a tag that would allow for a one-step detection; hence, we will conjugate a His-tag and a fluorophore (mNeonGreen), for simple purification, and detection of the recombinant antibodies. mNeonGreen is a brighter fluorophore than the commonly used Green Fluorescence Protein (GFP), which may allow for a more sensitive assay. The engineered constructs will be ordered as gene fragments and cloned and expressed in E. coli.

Sources

1. Gjerde, H., Clausen, G. B., Andreassen, E., and Furuhaugen, H. (2018) Evaluation of Dräger DrugTest 5000 in a Naturalistic Setting. J Anal Toxicol. 42, 248–254
2. Niemi, M. H., Turunen, L., Pulli, T., Nevanen, T. K., Höyhtyä, M., Söderlund, H., Rouvinen, J., and Takkinen, K. (2010) A Structural Insight into the Molecular Recognition of a (−)-Δ9-Tetrahydrocannabinol and the Development of a Sensitive, One-Step, Homogeneous Immunocomplex-Based Assay for Its Detection. Journal of Molecular Biology. 400, 803–814
3. Brennan, J. (2005) The production of recombinant single chain antibody fragments for the detection of illicit drug residues. doctoral thesis, Dublin City University, [online] http://doras.dcu.ie/17319/ (Accessed March 12, 2019)