Investigation of interactions between nanobodies and their antigens using SPR detection methods

Examensarbete för masterexamen
Master Thesis
Mariotti, Michele
Nanobodies, or single domain antibodies, have gained a lot of attention recently in nanoscience because of their attractive properties such as small size, high stability, high flexibility, hydophilicity and ease of manufacture, which make them ideal candidates for a lot of application areas, including biotechnology, therapeutics and diagnostics. The aim of this Master Thesis is to evaluate the interactions between nanobodies and their binding partners by Surface Plasmon Resonance techniques. The goal of the first part of the thesis is to overexpress, purify and characterize three different proteins: His-eGFP, GFP and nanobody. The results indicated a succesfull overexpression and purification of His-eGPP and GFP, with a high level of purity achieved for both proteins. Nanobody was overexpressed but its purification led to many challenges, as the final purified protein had a very low density. The second part of the thesis investigates the experiments aimed at evaluating the interactions between a nanobody against GFP and a GFP protein, previously purified, on two biosensor surfaces: a Ni:NTA chip and an anti-his antibody immobilized on a CM5 chip. Furthermore, the interaction of a single nanobody on the two surfaces is compared with His-eGFP interaction on both surfaces. Results showed that the interaction between nanobodies and their binding partner can be investigated by SPR. In particular, nanobody showed a higher affinity for both biosensor surfaces compared to His-eGFP. Kinetics measurements indicated higher association rate constants and lower dissociation rate constants for nanobody binding to the anti-his-antibody surface compared to His-eGFP binding to same surface, confirming the high affinity of the nanobody for the anti-his-antibody surface. Results of the experiments involving the interaction between GFP binding to nanobody immobilized on the two surfaces indicated a very similar affinity at equilibrium of GFP for the immobilized Nb on both surface. On the other hand, kinetics measurements showed that GFP had a higher affinity for nanobody immobilized on the anti-his surface, as slower dissociation rate constants and slightly higher association rate values were obtained. In conclusion, the interaction between nanobody and its antigen was succesfully characterized and evaluated by SPR, but care must be taken while choosing the experimental parameters in order to minimize mass transport problems typical of SPR experiments.
Livsvetenskaper, Biologiska vetenskaper, Bioinformatik och systembiologi, Medicinsk bioteknologi, Life Science, Biological Sciences, Bioinformatics and Systems Biology, Medical Biotechnology
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