Abstract
Single domain antibodies are recombinantly expressed functional antibodies devoid of light chains. These binding elements are derived from heavy chain antibodies found in camelids and offer several distinctive properties for applications in biotechnology such as small size, stability, solubility, and expression in high yields. In this study we demonstrated the potential of using single domain antibodies as capturing molecules in biosensing applications. Single domain antibodies raised against green fluorescent protein were anchored onto biosensor surfaces by using several immobilization strategies based on Ni2+:nitrilotriacetic acid-polyhistidine tag, antibody-antigen, biotin-streptavidin interactions and amine-coupling chemistry. The interaction with the specific target of the single domain antibodies was characterized by surface plasmon resonance. The immobilized single domain antibodies show high affinities for their antigens with KD = 3–6 nM and outperform other antibody partners as capturing molecules facilitating also the data analysis. Furthermore they offer high resistance and stability to a wide range of denaturing agents. These unique biophysical properties and the production of novel single domain antibodies against affinity tags make them particularly attractive for use in biosensing and diagnostic assays.
Highlights
Single domain antibodies, referred to as nanobodies (Ablynx) or VhH, were discovered in the serum of camelids by Hamers-Casterman and co-workers in 1993 [1]
HEPES, NaCl, EDTA, tween 20, NiCl2, glycine, biotinamidohexanoic acid N-hydroxysuccinimide ester (Bt-NHS) and all the materials used for protein expression and purification were purchased from Sigma-Aldrich (Denmark). 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ethanolamine-HCl, sensor chips CM5, nitrilotriacetic acid (NTA) and CAP and monoclonal anti-polyhistidine antibody were from GE Healthcare (Denmark)
Single domain antibodies against Green Fluorescent Protein (GFP) presenting a six histidine-tag at the C-terminal were obtained from Chromotek GmbH (Germany) as GFP-Trap, biotin-labelled monoclonal anti-GFP antibodies were from Novus Biologicals (Denmark), monoclonal anti-GFP antibodies were from Invitrogen (Denmark)
Summary
Referred to as nanobodies (Ablynx) or VhH, were discovered in the serum of camelids by Hamers-Casterman and co-workers in 1993 [1]. They represent a unique type of functional antibodies that lack the light chains, while preserving the antigenbinding properties of conventional antibodies. Single domain antibodies are resistant to extreme pH, heat denaturation, proteolysis, solvents and detergents [12,13,14,15] They can be produced in high expression yields in heterologous systems such as bacteria or yeast and are very easy to purify and handle [11]. Upon immobilization, single domain antibodies might present different properties from the ones observed in solution [16] and their potential as capturing agents still needs to be characterized in details
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
