The debate regarding whether monoclonal antibody reagents are better than polyclonals has been raging for years. While many researchers praise the batch-to-batch consistency and single-isotype nature of monoclonals, others swear by the ability of polyclonals to work in a wider range of applications, often enabling detection of the target antigen in both its native and denatured states. Although these arguments remain valid, the well-publicized reproducibility crisis has fueled the discussion, encouraging a growing trend toward the use of antibodies produced using recombinant technology, and taking the conversation in new direction altogether.
Remaining strongly in favor of polyclonal antibodies, Shaad Farooqui, product development manager at FabGennix, explains that with reactivity across a broad range of species and the capacity to bind multiple epitopes, polyclonals represent a research tool that has stood the test of time. “Irrespective of clonality, the biggest issues with antibodies these days are a lack of specificity and poor reproducibility,” he says. “These are exacerbated by the distributor model many major antibody companies employ. To combat this, we adhere to Single Source Antibody™ manufacturing within FabGennix, exclusively selling antibodies generated and characterized in house. By performing every stage of the process ourselves, we can ensure better product quality through tighter control.”
Also highlighting the advantage of multiple epitope recognition, Sian Bolitho, product manager at Biorbyt, suggests that polyclonals represent a cost-effective option when a researcher is just starting to work with a target and hasn’t yet optimized a protocol. “Beginning assay development with a polyclonal maximizes the potential to return a good signal due to the fact these antibodies bind multiple sites on a target protein,” she says, “however, the choice between monoclonal or polyclonal will ultimately be governed by the general aims of the experiment. A researcher developing an ELISA may choose to use a polyclonal for capture and a monoclonal for detection to maximize specific signal, whereas a researcher producing a diagnostic lateral flow assay is likely to employ a monoclonal to afford consistency over an extended period.”
Bolitho adds that a further consideration is the amount of antibody required. “With polyclonals, production is limited by the size of the immunized animal. In contrast, monoclonals can be produced in much larger quantities. Imagine the amount of antibody required for common lateral flow assays such as pregnancy tests, and you can clearly see why industrial-scale monoclonal antibody production methods such as wave bag systems or fermenters have been developed.”
Falling more on the side of monoclonals, Dr. Michael Weiner, vice president, molecular sciences at Abcam, points out that recent data from CiteAb indicates the use of monoclonal antibodies to be growing at a faster rate compared to polyclonals, a trend he believes will continue over the next few years. “One reason for the increased use of monoclonals could be that many journals are heavily promoting the use of more reproducible reagents,” he says. “An additional driver may be the ongoing commitment across the industry to reducing animal usage. Polyclonal antibody production involves repeated immunization of animals, whereas monoclonal production requires just one round. Importantly, however, recombinant antibody technology replaces the need for animal use entirely.”
Recombinant technology
Abcam have been adding recombinant monoclonal antibodies to their portfolio since 2012, and today make all their new products recombinantly. “By engineering recombinant versions of our popular RabMAb® rabbit monoclonals, we’ve been able to build a portfolio of over 13,000 recombinant monoclonal antibodies to date,” reports Weiner.
“The rabbit immune system generates antibody diversity and optimizes affinity by mechanisms that are more efficient than those of rodents, and it’s also better for producing antibodies against small molecules and peptides that do not elicit a good immune response in mice. By combining these advantages with recombinant technology, we can improve both antibody specificity and sensitivity, while significantly shortening timelines for antibody production and eliminating animal use. A further benefit is that, once sequenced, recombinant monoclonals are not subject to loss of activity caused by sequence drift, or loss of yield, both of which can affect hybridomas if they are poorly maintained.”
“Monoclonal antibodies are great,” notes Michael Fiebig, business development director for products and innovations at Absolute Antibody, “however we must not forget that monoclonals are traditionally produced by hybridomas—the result of fusing antibody cells with myelomas. Myelomas often contain additional antibody transcripts, diluting the amount of ‘active’ antibody in your vial; moreover, they are cancerous. Genetic instability, one of the hallmarks of cancer, is inherited by hybridomas, and can lead to loss of activity, isotype switching and reduced expression.”
Earlier this year, Absolute Antibody co-authored a publication challenging the perception that all monoclonals are monospecific and identical from lot-to-lot. Within this it was reported that around a third of hybridomas make extra light-chain and heavy-chain protein—the additional antibody transcripts just mentioned. “Under different culture conditions, you will get different relative abundances of these transcripts and your batch-to-batch reproducibility will fluctuate greatly,” explains Fiebig. “If you’re doing a Western blot, maybe you won’t be too worried if you need to dilute your monoclonal antibody 1:1000 versus 1:300, but once you start considering more quantitative research methods or the entire diagnostics industry, this can cause huge problems.”
Image: Many forms of antibody engineering can be achieved using recombinant technology. Image courtesy of Absolute Antibody.
Offering only recombinant antibodies within their portfolio, Absolute Antibody firmly believe that recombinant technology will shape future antibody development. “Recombinant antibodies give you the certainty of working with a precisely defined product, and I would recommend that a researcher always use a recombinant antibody wherever possible,” says Fiebig. “From a cost-perspective, most recombinant versions of classic clones are the same price, if not cheaper, than the hybridoma-produced version, so antibody selection is really just a case of changing habits. We need to stop wasting time re-optimizing protocols and re-calibrating assays, and accept that antibodies need no longer be the bottleneck in studies because of something as simple as the format they’re in.”
With recombinant antibody technology opening up so many new possibilities, the opportunity for researchers to request a product exhibiting specific properties is becoming increasingly accessible. “As we develop innovative ways of engineering antibodies, we can insert desirable features during the cloning steps,” reports Weiner. “For example, if we want to increase the thermostability of an antibody, we can easily do so by cloning in the right gene sequence to make this happen.”
Fiebig adds that while using recombinant antibodies will likely one day become standard practice, Absolute Antibody are currently focused on areas where antibody engineering opens up obvious benefits. These include in vivo research—matching antibody to target species; virology and allergy research—specific human and mouse IgG, IgM, IgE; anti-tag antibodies—new versions to provide more options for secondary antibodies; and isotype controls—one specificity in every format to streamline assays. “We’re seeing more and more people embrace recombinant antibodies to take their research to the next level,” he says. “The past few years have seen a great change in awareness of, and appreciation for, recombinant antibody technology, and this is definitely continuing to grow.”