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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Tags: affinity reagents, capture molecules, human proteomics, IgG, nAb: Camelid Antibodies, nAbs, nano antibodies, nanobodies, proteome, scFv, VHH, VHH antibody chromotek

BioTechniques Publishes Article on Single Domain Antibodies

Many blogs start by asking “Did you know…” to intrigue you to read along. So here it goes:

Did you know that there are more than 300,000 antibodies that are commercially available? And yes, many antibody companies are still generating more antibodies at ever faster pace and in a more systematic way. There are companies that plan to make peptide or short protein fragments for making antibodies against all human proteins or subproteome, others develop antibodies particularly suitable for demanding assays such as ChIP-CHIP. Government activities such as the National Cancer Institute (NCI)’s Clinical Proteomic Technologies Initiative (CPTI) and the Road Map program under the NIH Director’s Office also set goals of producing comprehensive sets of widely usable, renewable, affinity reagents for clinical cancer samples or the human proteome. Apparently people do not think the 300,000 available antibodies are sufficient for what they do.

Did you know that conventional antibodies commonly used as reagents are ~150kDa in molecular weight and can hardly be used inside live cells? Ulrich Rothbauer, professor in the department of biology at Ludwig Maximilians University, who is working with colleagues to develop tools to study cellular processes in living cells. “These antibodies have to assemble four different chains, two heavy and two light, and they’re assembled by disulfide bonds that cannot be correctly formed in the reducing environment of the cytoplasm. You cannot express such a huge complex molecule in living cells. You can [introduce] them by microinjection, for example, but it’s not applicable for high-throughput cell imaging.” [1] Antibody fragments such as scFv, Fab, and similar derivatives have been developed over the years to certain level of success, but not as widely accepted or practically amenable to replacing conventional antibodies.

Did you know that camel, llama, and shark naturally produce single heavy chain antibodies that can function as 13-16kDa fragments (yes if you have read previous Allele Blogs http://allelebiotech.com/blogs/2009/08/camelid-antibodies/)? They can easily be produced in bacteria, used directly inside live cells via transgene, fused to other proteins as a fusion tag, linked to DNA oligos as a detection module, or immobilized on beads for pull down or co-IP. Currently, these antibodies need to be selected by display after obtaining immunized antibody libraries. There is generally no commercial service for creating custom camelid antibodies at this time due to patent and other issues. Existing products are available for jelly fish GFP and DsRed derived RFP fusions. Publications using such a limited number of camelid antibodies have been amazing so far—dozens in top journals within the last few months and after only a short period of time since product launch.

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Original BioTechniques Article http://www.biotechniques.com/news/biotechniquesNews/biotechniques-257771.html?utm_source=BioTechniques+Newsletters+%2526+e-Alerts&utm_campaign=b94f127de0-Methods+Newsletter&utm_medium=email

Tags: Alpaca, camelid antibodies, Chromotek, Fab, GFP-Trap, Hc antibodies, mCherry, mOrange, mPlum, mRFP1, mRuby, nAbs, RFP-Trap, scFv, single domain antibodies, VHH, VHH antibodies