By now, most of us are familiar with the 2015 Nature publication calling for standardization of antibodies used in research. Signed by over 100 eminent scientists worldwide, this proposed that antibody reagents should be produced recombinantly to avoid wasting time, money, and samples due to experimental findings being impossible to replicate. Not only do recombinant approaches to antibody production promise to improve the reproducibility of any immunoassay technique, but they also allow researchers to explore new experimental designs. This article looks at some of the ways recombinant antibodies are driving research.
Defining recombinant antibodies
According to Dr. Michael Fiebig, VP product portfolio and innovation at Absolute Antibody, we should be careful how we define recombinant antibodies. “It is important to differentiate between an antibody discovered by recombinant methods such as phage- or yeast-display, and an antibody produced by recombinant methods,” he says, noting that producing antibodies recombinantly includes the former as well as obtaining antibodies by sequencing hybridomas.
“Antibodies discovered by recombinant methods require additional validation as they do not benefit from the tolerizing effect of an immune system, generally preventing binding to common protein patterns in a mammalian host,” he explains. “Moreover, antibodies that may transition from an in vitro to an in vivo assay need to be screened for activity and stability under physiological conditions, especially temperature—something that is an inherent property of an antibody made in a mammal—unless the selection/panning process has been set up with this in mind. Additionally, it is important to consider properties beyond the biological activity of the antibody if it is to be a viable resource for research. Just because you can make 50 µg of an scFv in a bacterial system and perform sufficient testing with it does not mean you will be able to manufacture hundreds or thousands of mg of whole antibody in a mammalian expression system. One must keep in mind that additional screening, and possibly protein engineering, is required to ensure that display-derived binders can reach their full potential.”
Antibody production against challenging targets
Katie Crosby, director, IHC at Cell Signaling Technology, notes that a valuable characteristic of animal-free approaches like phage-display is that they allow antibody development against targets that may be toxic to an animal or that are not highly immunogenic. “Until relatively recently, phage display was a costly, labor-intensive approach to antibody discovery that was used predominantly to develop biologics,” she says. “Now, this powerful in vitro method allows for the rapid production of recombinant antibodies for research that would be difficult or impossible to generate using animals.”
Streamlining the discovery of effective antibody pairs
One way that recombinant technology has proven beneficial is in guaranteeing a renewable supply of antibodies that can be combined as matched antibody pairs. “It is challenging to find two antibodies against the same target that recognize different epitopes and can be combined as a pair,” reports Dr. Alejandra Solache, VP new product development at Abcam, “yet identifying such pairings is vital to support immunoassays and multiplexing. Recombinant rabbit monoclonal technology offers a significant advantage when it comes to generating antibody pairs that can work together in the same assay.”
Building on this point, Solache notes that while developing antibody pairs in mice is difficult due to the limited immune repertoire, the rabbit immune system uses somatic gene conversion and somatic hypermutation to produce a diverse B-cell population that provides a wider range of antibodies. “Rabbit antibodies are better than rodent antibodies at distinguishing subtle differences such as epitope variations, PTMs and conformational changes, or at detecting small molecules,” she says. “They also have a higher affinity than most mouse monoclonals for a target antigen. By immunizing rabbits and converting the resulting hybridoma-produced antibodies to a recombinant format, we have been able to secure a consistent source of highly specific recombinant RabMAb® monoclonal antibodies. These power many of the immunoassays based on our Matched Antibody Pairs, including SimpleStep ELISAs and FirePlex immunoassays, and give scientists confidence that assay performance will not be adversely affected by the presence of additional antibody heavy and light chains that can occasionally be present in hybridoma-produced monoclonal antibodies.”
Image: Recombinant antibodies provide exceptional batch-to-batch consistency, as demonstrated by testing data for multiple batches of Abcam’s recombinant RabMAb® anti PD L1 antibody [28 8] (ab205921) that were produced over a 4-year period.
Increasing the scope of existing applications
Another way that recombinant technology has shown its worth is in overcoming researchers’ dependence on full-length antibodies. “I think especially in highly sensitive ELISAs or in Lateral Flow Immuno-Assays (LFA), the ability to create antibody fragments recombinantly is proving a very popular option,” reports Fiebig. “In some sandwich ELISAs, using monomeric Fab fragments can help improve the dynamic range by maintaining a 1:1 stoichiometry between analyte and detecting antibody. Moreover, when Fabs or Fab2s are used as capture antibodies in ELISAs or coated onto particles in LFAs, a much higher density of antigen-binding sites can be achieved than when using full-length antibodies, where the Fc domain takes up a lot of space with no antigen-binding capacity.”
Fiebig adds that the site-specific functionalization of Fab fragments (also applicable to Fab2 or full-length antibodies) further highlights the utility of recombinant technology. “Genetically creating specific conjugation sites, or adding tags that can be functionalized, allows for the creation of site-specific labeling sites and more tightly controlled labeling stoichiometries,” he says. “Such modifications can also be used to improve directional immobilization, so that more of the business end of the antibody is facing the right way, rather than a random coating of the antibody onto a surface.”
Supporting in vitro to in vivo transition
Recombinant technology has also been important to smooth the transition from in vitro to in vivo research. “Recombinant rabbit monoclonals are particularly valuable when transitioning from a cell-based assay to an ex vivo tissue-based assay,” notes Solache. “Since animal studies are predominantly carried out in mice, using recombinant rabbit monoclonals avoids the issue of mouse-on-mouse cross-reactivity.” She adds that a major advantage of recombinant antibodies in general is that because the DNA sequence is known, there are multiple ways to optimize and adapt recombinant antibodies for further studies.
“With recombinant antibodies, the same antigen-binding domains may be employed in the context of different Fc domains to reduce the occurrence of neutralizing antibodies and undesirable adverse immune reactions when the antibody is injected into an animal,” explains Fiebig. “Not only does this increase the timeframe within which in vivo studies can be performed, but it also gives researchers the possibility to choose the desired antibody effector function. This is important not just for animal welfare but also for ensuring the best-possible experimental reproducibility as the dynamics of the induction of neutralizing antibodies is taken out of the equation as well as poorly characterized cross-species antibody effector function.” To aid the transition to in vivo research with syngeneic antibodies, Absolute Antibody recently launched the VivopureX™ range, where they have taken popular antibody clones, many originally obtained from rats or hamsters, and engineered them into mouse-anti-mouse recombinant versions. “These chimeric antibodies allow researchers to use the same antigen-binding domain with an engineered species-matched Fc domain, improving research results in mouse models through reduced immunogenicity, and increased long-term efficacy and potency,” says Fiebig.
Thorough antibody validation remains essential
Regardless of the means of production, all antibodies should be carefully validated. “Recombinant expression is not subject to the common pitfalls associated with hybridomas, like gene loss or cell line drift, for example, meaning that antibodies expressed recombinantly perform quite consistently from lot-to-lot,” reports Crosby. “However, an antibody should always be validated for its performance in the intended application, and it is sensible to confirm that antibody behavior remains consistent as new lots are introduced into the lab.”
Solache summarizes that while we must clearly distinguish between how an antibody is made and how it is validated, once you have a well-characterized antibody, the ability to make it consistently from lot-to-lot—as is possible with a recombinant clone—is a huge improvement over hybridoma-developed monoclonals and particularly over polyclonal antibodies. “Interestingly, data from our extensive KO validation testing contradicts the traditional assumption that top-cited antibodies are more likely to be ‘the best’ or that they will have the highest specificity to the target of interest,” she says. “We’ve observed that a significant number of top-cited antibodies actually display off-target effects. This strengthens the case for life scientists to use recombinant monoclonal antibodies that have been validated for specificity to their target whenever suitable clones are available.”
The importance of recombinant antibodies in flow cytometry
Miltenyi Biotec recently asked six experts from the flow cytometry and antibody community—including Andrew Bradbury, Ph.D., CSO of Specifica, who published the 2015 Nature publication—to share their opinion and experience with recombinant antibodies. This is summarized in a series of short videos and includes insight from:
- Dr. Anis Larbi, head of the flow cytometry platform at the A*STAR Institute in Singapore, who talks about the advantages of recombinant antibodies in terms of background signal
- Dr. Joffrey de Larichaudy, analytical development manager at CELLforCURE, who explains the value of recombinant antibodies to improve panel precision and limit variability
- Dr. Karl Johan Malmberg, professor of immunology at the Oslo University Hospital, who describes how recombinant antibodies are helping to advance natural killer (NK) cell research