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(859) 323-9770 jsblackburn@uky.edu

New Methods for Examining Nanobody Specificity and Binding to PRL-3

At our last lab meeting, I updated everyone on my project that has thus far focused on designing nanobodies, derived from alpaca heavy-chain only antibodies, that specifically bind to PRL-3. One of the biggest caveats to studying PRL-3 is the lack of tools that are currently on the market that directly target this protein. We decided to take it upon ourselves to start designing our own tools, in order to have antibodies that specifically target PRL-3 over PRL-1 and PRL-2 that can be used in a variety of assays.

Thus far, I have managed to clearly demonstrate that our nanobodies are specific for PRL-3 in vitro and in cellular models (C.N. Smith, et al., 2020 bioRxiv). Our new, and quite exciting, data has now allowed us to define specifically which nanobodies bind to PRL-3 with the greatest affinity using Biolayer Interferometry. This technique measures binding to your antibody of choice as a function of light, where we were able to bind our nanobody to a sensor then measure PRl-3 binding with increasing concentrations of protein to calculate a global Equilibrium Dissociation Constant (KD) for each nanobody. Every nanobody we tested has a binding affinity for PRL-3 in the nanomolar range, making them quite comparable to antibodies currently on the market and used in clinic.

Our next steps include deciphering the binding site for a subset of our nanobodies on PRL-3. We will utilize multiple approaches, including Hydrogen Deuterium Exchange Mass Spectromotry (HDX-MS), with our collaborators Daniel Deredge and Kyle Kihn at the University of Maryland School of Pharmacy. We will also attempt co-crystallization of PRL-3 and nanobodies using X-ray Crystallography methods with the help of members of the UK Molecular and Cellular Biochemistry Structural Biology Core.

Biolayer Interferometry (BLI) was completed using ForteBio’s BLItz technology. BLI is a technique that allows for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of the biosensor tip.

 

Caroline Smith is a fifth year PhD candidate in the Department of Molecular and Cellular Biochemistry.

 

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