Binding Decisions: Designing the Optimal Custom Antibody

 Custom Antibody Production

Antibodies are the workhorses of the immune system, detecting and neutralizing attacks from foreign antigens. Researchers have also harnessed antibodies’ ability to bind to specific antigens to create powerful molecular-detection devices, enabling scientists to probe for particular diagnostic markers, infectious agents or other targets.

If you’re working in any kind of molecular analysis, chances are you’ll be taking advantage of antibody technology of one sort or another. Often you’ll find what you need by flicking through a product catalog (see Biocompare's antibody search tool here), but sometimes an off-the-shelf antibody solution doesn’t quite fit the bill. When that’s the case, a custom antibody-production service may be your best option.

“While the listings of commercially antibodies may seem endless, there are many reasons a scientist would choose the custom-production route,” says Neal Kitchen, product manager for immunoassays at Thermo Fisher Scientific. “For example, new antibodies need to be developed, because we are still discovering proteins.” Commercially available antibodies may also lack specificity or functionality in the application a researcher has in mind, whether that be Western blottingimmunohistochemistry or chromatin immunoprecipitation.

And then there are the very active areas of research looking at post-translational modification of proteins. “In many instances, a researcher wants to track not only the presence or absence of a protein but also to be able to interrogate post-translational modifications on that protein that affect its biological activity in a given pathway,” explains Tom Turi, vice president, science and technology, discovery and translational services at Covance. In those situations, researchers can also turn to custom-antibody services.

Things to consider

So what do you need to think about if you’re going down the custom route? Broadly, your initial choice is between polyclonal and monoclonal antibodies. Polyclonal antibodies are purified from serum from a host animal that has been immunized with the antigen of interest. This will get you a collection of antibodies from different B cells, each recognizing different epitopes on the same antigen. Monoclonal antibodies, on the other hand, are antibodies from a single B cell that bind only to a single epitope. Because B cells are relatively short-lived, they are generally fused with myeloma cells to produce immortal hybridoma cell lines that express the desired antibody.

Both approaches have advantages and disadvantages, depending on your specific needs.

Polyclonal antibodies are cheaper and faster; they can cost as little as $500 or $1,000, for a vanilla product, to about $3,500 or so for more complicated projects that require multiple purification steps, says David Chimento, assistant laboratory director at Rockland Immunochemicals. Monoclonal projects are more expensive, costing $8,000 to $25,000, but yield a much more robust reagent at the end.

Polyclonal projects are also faster. They can be carried out in as little as 45 days for immunization and initial screening, with extra time for additional purification steps, whereas with monoclonal projects you’re likely to be looking at 100 days or more.

Quality and quantity

Of course, time and cost are irrelevant if the antibody doesn’t perform as you need. “When you're generating an antibody, it's good to have the end assay in mind,” says Chimento. “And it should be up and running at the time you start the project, such that you are able to properly screen.”

“One of the most important factors to consider in the decision process is the application for which the antibody is used,” agrees Turi. “For example, in biotherapeutic development, a positive control for antidrug antibody is in most cases a polyclonal antibody, because one wants to have antibodies against all possible epitopes.” On the other hand, when identifying a post-translational modification you want an antibody that recognizes only the modification itself (in its protein context), in which case a monoclonal antibody is the logical choice.

In general, polyclonal antibody production is better for qualitative detection methods like Western blots, and monoclonal antibodies may be more appropriate in quantitative assays such as ELISA. If you’re drawn to polyclonal antibodies for reasons of time and cost, but you need the specificity offered by monoclonal antibodies, “monospecific" antibodies might prove useful (these are polyclonal antibodies that have been put through an additional purification step, so they approach the specificity of monoclonal antibodies). Rockland Immunochemicals, for example, offers affinity purification and cross-absorption processes for polyclonal antibodies to boost their specificity. Other firms, such as Pacific Immunology and Thermo Fisher Scientific, provide similar custom monospecific-antibody production services.

There also is the question of whether to generate antibodies against a full-length protein or a peptide. Generating antibodies against the full protein will produce a selection of antibodies against a number of different epitopes, thus maximizing the chances of recognizing the protein in the target assay but also increasing the chances that you’ll pick up other nontarget proteins. In contrast, using a peptide sequence will generate antibodies that are highly specific to your target protein (for example, antibodies against point mutations, polymorphisms or post-translational modifications). But the potential downside to that strategy is that the chosen sequence might not correspond to an exposed region of the native protein, so you have to try and accurately predict the location of peptide sequences within the full protein structure.

More or less

Another key consideration is supply. “The key difference with monoclonal antibodies: Once you generate it, you have an indefinite supply of that reagent,” explains Chimento. This is important if you need a robust antibody that you’ll be using for an extended period of time, if you’re planning to commercialize the antibody or use it in some kind of diagnostic or therapeutic application, for example.

Similarly, once you establish culture and purification conditions, you can anticipate that monoclonals will be relatively consistent and homogeneous from lot to lot. Polyclonals, though, are more variable. “They are heterogeneous, and the quantity is limited by the size of the host species and its life span,” explains Turi. “In addition, polyclonal antibodies generated to the same antigen in multiple animals differ among immunized animals.”

But polyclonal antibodies may also be a better option in some cases. If your target protein is only expressed in low levels, for example, polyclonal antibodies will help amplify that signal as multiple antibodies bind to different epitopes on the protein. “Having multiple points of detection usually offers a more robust detection in applications like Western blotting, immunoprecipitation and chromatin immunoprecipitation,” says Kitchen. “And a good polyclonal antibody can have great sensitivity to the target in many applications.”

One final consideration involves more specialized antibody designs. In general, researchers will use an entire antibody molecule in their work. Sometimes, though, it is useful to remove the parts of the molecule that aren’t actually needed for binding a target antigen and use just a fragment of the molecule. Several firms, for example, offer custom production of Fab and single-chain variable fragments (scFv); these are smaller, so they may be able to access a target more effectively than full antibodies, and they have been shown to have enhanced functionality in certain immunoassays.
 
Ultimately, your goal in pursuing custom antibody production is to obtain an antibody that performs well in your intended application. Custom antibodies are costly to be sure. But for some users, there may be benefits that can’t be matched by a commercially available antibody.

“For custom production, companies such as Thermo Fisher Scientific give full ownership to the customer,” explains Kitchen. “Since the customer will own all of the material, it can be invaluable to the project and in some cases provide the end user an opportunity to commercially license the antibody and gain long-term royalties—especially if the antibody is the ‘best on the market.’”

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