Antibody Production

Editorial Article

 

by Caitlin Smith

  

October 04, 2011

  

Identifying proteins – whether in Western blots or live-cell fluorescent imaging – almost invariably requires an antibody, a protein that binds specifically to one protein or group of proteins. Not all antibodies are alike, however. Monoclonal antibodies are designed to be highly specific to one protein target, whereas polyclonal antibodies are less specific, but also have more flexibility in their uses. Polyclonal antibodies are also faster and less expensive to produce. Whether you construct the antibodies yourself or purchase them from an antibody production service, here are some points to consider when choosing monoclonal and polyclonal antibodies.

 

General considerations

The antigen
Choosing an antigen – the peptide or protein that the antibody will target – is one of the first steps in antibody generation. “Starting off with the antigen against which antibodies are to be generated,” says Thomas Kohl, assay development group leader at Rockland Immunochemicals, “the researcher needs to ensure quantity, stability, and purity of the antigen to be used.” The characteristics of the antigen employed can affect the antibody production process. “Are you going to be using a recombinant protein or a synthetic peptide to raise the antibody?” asks Cecilia Po, technical support and marketing specialist for Anaspec. “If using a peptide, then one should consider whether the sequence has good surface probability and solubility in an aqueous environment. Such peptides are more likely to be antigenic. A hydrophobic sequence may pose technical difficulties in peptide purification, carrier protein conjugation, and resin coupling for antibody affinity purification.”

 

Time, specificity and reproduction
Monoclonal antibodies may take 4-5 months to generate, whereas polyclonal antibodies can take as little as one month. And of course, specificity is key. “Monoclonal antibodies recognize a single epitope of the target protein or peptide antigen, and the epitope can be defined,” says Po. “Also, monoclonal antibodies can be easily reproduced. Polyclonal antibodies recognize multiple epitopes of the target protein or peptide antigen, and the epitopes are not easily defined. In general, the polyclonal antibody activity is animal-specific, and therefore not easily reproduced.”

Further and more detailed considerations for each type of antibody, along with some current offerings from vendors in these areas, can help you make the best choice for your needs.

 

Monoclonal antibody production, key considerations:

Time and expertise
The reason monoclonal antibodies take longer to make is that they use a more advanced technology based on cultured hybridoma cells. Taking care of these cells results is a more expensive and time-consuming process.

Thermo Fisher Scientific offers a custom antibody production service (for both monoclonal and polyclonal antibodies), as well as “do-it-yourself” products. “Our custom service uses Thermo Scientific Antigen Profile Software, an advanced antigen design program with powerful algorithms that guarantee the best peptide antigen design,” says Neal Kitchen, product manager for Thermo Scientific immunoassay products. “Once the antigen is designed, we use a novel technology for antigen presentation called Thermo Scientific Targeted Antigen Display (TAD) Technology, which allows multiple conformations of the antigen. This technology is extremely important to generating functional antibodies over multiple applications.” They also offer a range of antibody production and purification reagents for doing it yourself. These include carrier protein kits, immunogen preparation reagents, isotyping kits, antibody purification resins and immunoglobulin fragmentation kits. “Our newest purification products include our Thermo Scientific Easy-Titer Antibody Assay Kits, and Thermo Scientific Pierce Rapid Mouse Antibody Isotyping Kits, which allow researchers to confidently determine the concentration and isotype of their antibodies,” says Kitchen.

Thermo Fisher Scientific also offers a range of tools for large-scale production of antibodies made from cultured cells. “This employs bioreactor-based culture methods in such platforms as hybridoma and transfectoma,” says William Whitford, senior market manager in cell culture for Thermo Scientific cell-culture and bioprocessing products. “Single-use technology (SUT) for mixing, transport and storage of process materials, as well as in fully controlled bioreactors is now standard in the production of protein biologicals, including monoclonal antibodies. We provide a number of product types employing SUT, such as our BioProcess Containers for bulk material and product storage, cryopreservation, and waist containment, Single Use Mixers for process fluid preparation and mixing, and Single Use Bioreactors for large-scale animal cell culture.”

 

Specificity
For some applications, the greater specificity of monoclonal antibodies make them the only way to go. But there are other advantages to monoclonals as well, such as better reproducibility. “Researchers frequently look for monoclonal antibodies because of their specificity and generally lower background,” says Kitchen. “Because monoclonal antibodies come from a single clone, they have a much higher homogeneity than the heterogeneous mix from a polyclonal antibody. This feature can result in higher reproducibility from one experiment to the next, as well as comparable binding efficiency from one sample to the next. Moreover, once a successful hybridoma is produced, the researcher has a renewable source of antibody with nearly identical batch production, whereas a polyclonal can vary significantly from batch to batch.”

 

New technology on the horizon
If you decide you need to use monoclonal antibodies, but don’t want to wait out the lengthy production process, check out the new technologies being developed for monoclonal production. For example, Rockland Immunochemicals has developed a new myeloma tool to produce monoclonals more efficiently. “[This new technology uses] splenocyte fusion by electrofusion rather than the traditionally implemented polyethylene glycol-based method,” says Kohl. “Linear alignment of fusion partner cells and harvested splenocytes within the electric field ensure a higher percentage of actual cell fusion. This, in turn, results in a greater number of mono, bi and polynucleated cells, thus significantly increasing the overall success rate of producing numerous hybridomas. The widespread implementation of recently developed technology in which antibody-specific genes are directly cloned out of harvested splenocytes would further improve and simplify the monoclonal antibody generation process.”

 

Polyclonal Antibody Production

Time and cost
If your experiment can tolerate somewhat less specificity than monoclonals provide, you may benefit greatly from using polyclonal antibodies – they’re less expensive, faster to make, and have multiple uses. “If a customer is building an assay that they plan to market, it may make sense to pay a premium to have an unlimited supply of monoclonal antibody,” says Scott Lewis, director of the antibody division at New England Peptide. “However, for pure research needs, you can’t beat the cost (polyclonal antibodies are 1/5 the cost of monoclonals) and time (polyclonal turnaround time is less than half of that of monoclonals) advantages of a polyclonal antibody, even when factoring in the inherent variability in a polyclonal response.”

There are a few select vendors that offer polyclonal antibodies in a month or less, such as the AnaSpec Speedy 28-day polyclonal antibody service. AnaSpec offers custom polyclonal antibodies in 28 days following the first immunization. “Compared to conventional protocols that take 70 days or longer, antibodies raised using our program are ready in 28 days,” says Cecilia Po. “The protocol employs a proprietary mixture of Freund’s free immune-stimulatory compounds and one of the animal bleeds is guaranteed to have a titer of 1:20,000 or better as measured by ELISA.” Similarly, Eton Bio offers 30-day express polyclonal antibody service.

 

Specificity and quality
You can also buy pre-made polyclonal antibodies, which seems faster and easier (as opposed to making your own, or using a custom antibody service). However, Lewis cautions against this in the name of specificity and quality. “The extra attention and care taken in the design and production of a custom polyclonal antibody often results in overall time and resource savings for investigators,” he says. “For example, research performed with a readily-available catalog antibody that gives background reaction to undesired proteins produces inferior results that requires more time to interpret and/or additional experiments to confirm. Contrastingly, a bioinformatic-vetted, site-specific and protein-selective polyclonal antibody can provide a ready source (often 10-20 mg of antibody, relative to a few micrograms of a catalog antibody) of predictable, reproducible, and readily-interpretable results that enhance research progress. Moreover, it can provide significant cost savings through optimized reagent and resource usage.”

Lewis explains that configuring the antibody specificity to meet your needs is more efficient in the long run, and can even be used to tailor your antibodies to recognize post-translational modifications. “Catalog antibodies are often made by injecting full proteins or large fragments of proteins,” he says. “This technique has its benefits, but researchers should understand the risk that it may lead to unwanted cross-reactivity to other proteins in the same family or hierarchy.” New England Peptide uses a method involving aldehyde-based conjugation chemistry that can be used to attach a peptide to a carrier protein and/or an affinity matrix. “Our standard conjugation is still maleimide-based, but in situations where an internal cysteine exists in the desired epitope, this is less than ideal,” says Lewis. “Using this method does not interfere with any naturally existing reactive species from the 20 common amino acids and is especially important for one of our specialties: the generation of antibodies to post-translational modifications. Because the available sequence for use around the modification site is so restricted, these situations tend to arise much more often. Also, all typical ‘work-arounds’ for this situation (substitution of a serine in place of the cysteine or truncating the sequence to avoid cysteine inclusion) can potentially negatively affect the specificity. The new chemistry alleviates all these concerns.”

 

Intended use
Depending on your intended use for the antibody, polyclonals may be a better choice precisely because they are less specific. “The heterogeneity of polyclonal antibodies can allow them to more readily bind to the protein regardless of conformational changes or modifications,” notes Kitchen. “Moreover, having multiple points of detection (i.e., multiple epitopes) usually offers a more robust detection in applications like Western blotting, immunoprecipitation, and chromatin-immunoprecipitation (ChIP), and a good polyclonal antibody can have great sensitivity to the target in many applications. In some cases, researchers look to polyclonal antibody production to increase cross-reactivity between species or homologous proteins.”

Although antibody production has seen great advances in recent years, Kitchen believes that improving antibody production depends on a better understanding of how antibodies interact with their antigens. “No matter how good the software and production lines, there is no guarantee the antibody will work the way the researcher wants,” says Kitchen. “The vast number of high-quality antibodies available show we have improved significantly in our understanding but, as with most areas of research, there is still new information to unfold and new uses to discover for antibody research. There is no question antibodies will continue to play a vital role in virtually all fields of science. Antibodies are one of the few tools of science that are used over all platforms of research.”

 

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