Format

Send to

Choose Destination
See comment in PubMed Commons below
Methods Mol Biol. 2012;911:417-29. doi: 10.1007/978-1-61779-968-6_25.

Characterization of single-domain antibodies with an engineered disulfide bond.

Author information

1
Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada. greg.hussack@nrc-cnrc.gc.ca

Abstract

Camelidae single-domain antibodies (VHHs) represent a unique class of emerging therapeutics. Similar to other recombinant antibody fragments (e.g., Fabs, scFvs), VHHs are amenable to library screening and selection, but benefit from superior intrinsic biophysical properties such as high refolding efficiency, high solubility, no tendency for aggregation, resistance to proteases and chemical denaturants, and high expression, making them ideal agents for antibody-based drug design. Despite these favorable biophysical characteristics, further improvements to VHH stability are desirable when considering applications in adverse environments like high heat, low humidity, pH extremes, and the acidic, protease-rich gastrointestinal tract. Recently, the introduction of a disulfide bond into the hydrophobic core of camelid VHHs increased antibody thermal and conformational stability. Here, we present additional protocols for characterizing the effects of the introduced disulfide bond on a panel of llama VHHs. Specifically, we employ mass spectrometry fingerprinting analysis of VHH peptides to confirm the presence of the introduced disulfide bond, size exclusion chromatography, and surface plasmon resonance to examine the effects on aggregation state and target affinity, and circular dichroism spectroscopy and protease digestion assays to assess the effects on thermal and proteolytic stability. The disulfide bond stabilization strategy can be incorporated into antibody library design and should lead to hyperstabilized single-domain antibodies (VHHs, VHs), and possibly Fabs and scFvs, if selection pressures such as denaturants or proteases are introduced during antibody selection.

PMID:
22886266
DOI:
10.1007/978-1-61779-968-6_25
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Springer
    Loading ...
    Support Center