Finding the Best Capture Reagents

As capture reagents, monoclonal antibodies are the most widely used reagents for specifically detecting and quantifying proteins due to their very high specificity. However, development of monoclonal antibodies is time-consuming and expensive. In addition, many antigens prove to be non-immunogenic or extremely toxic, and therefore cannot be used to generate antibodies in animals. Furthermore, the large size of monoclonal antibodies (150 kDa) may limit their use in cases where more than one binding reagent competes for space to recognize closely juxtaposed epitopes. These limitations could arguably be the biggest hurdles to using monoclonal antibodies as capture reagents for a systematic study of the complete human proteome or for clinical applications of advanced proteomics.

Therefore, alternative capture reagents with high specificity, high affinity, and flexible size and structure that can be easily and cost-effectively produced are urgently needed in order to accelerate proteomic research. Single-chain variable-fragment (scFv) antibodies have been commonly used as alternatives in this regard. scFv is comprised of only the light chain and heavy chain variable regions connected by a peptide linker and with a molecular weight of 27 kDa. Since scFv retains the antigen-binding site of the variable regions, it inherits the specificity of an intact antibody and affinity. In addition, scFv can be easily expressed in yeast or in E. coli with yields in milligrams per liter. scFv can be linked to Fc of desired species specificity and maintain binding properties. If necessary, there is also the option of converting scFv into other antibody formats such as Fab or full IgG by simple cloning steps. The converted antibodies can also be efficiently expressed and purified in yeast or E. coli.

More recently, single domain antibodies that exist in nature were discovered that can be as small as half the size of scFv, and judging from the available data, superior in binding capabilities to scFv or even traditional IgG antibodies. This type of affinity molecules, termed VHH isolated from camelid animals or nurse shark, can be highly expressed in E. coli, linked to a fluorescent protein marker, or chemically conjugated to HRP or other signal generating moieties through a one step reaction.

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    Wednesday, January 12th, 2011 nAb: Camelid Antibodies, Nanobodies, VHH

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