Flow cytometry is one of many immunoassay techniques that is evolving as a result of recombinant antibody technology. With exceptional reproducibility in terms of production yield and quality, and a vast capacity for engineering, recombinant antibodies have opened up new possibilities in panel design. This increased flexibility means that, whether used alone or in parallel with traditional monoclonals, recombinant antibodies are providing researchers with valuable new insights.
Recombinant antibody technology abrogates Fcγ receptor binding
One way that recombinant antibodies have benefited flow cytometry is by addressing the high background that frequently results from antibody binding to Fcγ receptors. According to Dr. Michael Fiebig, business development director, products and innovations at Absolute Antibody, this unwanted signal makes it difficult to discern different cell populations accurately and can lead to both false positive and false negative readouts. “Using recombinant technologies to abrogate FcγR binding has been a significant development within the flow cytometry field,” he reports.
“Researchers are aware that isotypes such as mouse IgG2a/b and rat IgG2b are notorious for high background staining of macrophages, monocytes, or B-cells, yet with a lack of viable alternatives they have had to resort to Fc blockers in attempts to prevent this. Now, using Fc Silent™ antibodies, there is no need to extend flow cytometry staining protocols with an Fc blocking step. Instead, researchers have access to improved versions of classic clones that feature a small set of mutations to prevent FcγR binding.”
Greater antibody purity provides more consistent fluorochrome conjugation
Another way that recombinant technology is improving flow cytometry experiments is through more reproducible batches of fluorochrome-conjugated antibodies. These reagents are widely used for flow, but variations in fluorescence/protein (F/P) ratio can affect the consistency of brightness and staining patterns between antibody lots. “Conjugation is a complex procedure with many potential pitfalls, and a frequent source of variability is hybridoma-derived antibodies,” explains Johannes Fleischer, global marketing campaign manager at Miltenyi Biotec.
“A recent study has demonstrated that >30% of hybridoma-derived monoclonals contain one or more additional productive heavy or light chains, which makes standardized conjugation processes challenging. As a result, researchers are often required to reoptimize staining conditions upon switching to a new antibody-fluorochrome batch. In contrast, our REAfinity™ recombinant antibodies are produced under standardized cell culture conditions and show excellent purity in MS analysis—this is the basis for more consistent conjugation.”
Image: REAfinity™ antibodies are defined products, while hybridoma-generated antibodies can be a mixture. MS analysis shows two examples of hybridoma-generated antibodies to each contain a second light chain (A); this is not present in REAfinity™ antibody products (B). Image courtesy of Miltenyi Biotec.
Recombinant antibody technology supports multiplexing
Recombinant antibodies have been pivotal to the development of increasingly complex flow cytometry panels. For example, using Abcam’s RabMAb® recombinant rabbit monoclonal antibodies, researchers can expand panel size and improve assay sensitivity without incurring unwanted background signal. “Flow cytometry usually relies on mouse, rat, and hamster antibodies,” notes Wayne Austin, Ph.D., senior scientist, flow cytometry at Abcam. “However, rabbits have a unique immune system that generates antibody diversity and optimizes affinity by more efficient mechanisms than the immune systems of rodents. By combining the high specificity and affinity of rabbit monoclonals with recombinant technologies, our RabMAb recombinant rabbit monoclonal antibodies provide greater capacity to detect low abundance targets, and subtle antigenic differences such as epitope variations, post-translational modifications, and conformational changes. Moreover, since the rabbit antibody sequence is distinct, RabMAb recombinant rabbit monoclonal antibodies are easily incorporated into staining mixtures to increase panel size without the risk of non-specific cross-reactivity.”
The degree of multiplexing can also be increased using recombinant technology to expand the diversity of species and isotypes within an antibody panel; this permits parallel staining with different secondaries. “In our experience, the ability to switch the Fc region to prevent clashes with other antibodies used in a flow cytometry panel has been very popular,” says Fiebig. “Where you may originally have been stuck with three mouse IgG1 antibodies, you can now use a mouse IgG1, a rat IgG1, and a rabbit IgG simultaneously. Conversely, for direct conjugates, you can push all your antibodies to have the same Fc region, thereby reducing the number of controls you need to purchase and have in stock at any one time; in this manner you need only a single isotype control antibody for each fluorophore.”
The onus is on manufacturers to ensure batch-to-batch consistency
“With recombinant antibodies becoming more widely perceived to offer greater reproducibility, there is a danger that researchers may view monoclonal antibodies generated from traditional hybridoma methods as inferior,” observes Kenta Yamamoto, product manager, flow cytometry at BioLegend. “This is not necessarily the case—numerous hybridoma-sourced antibodies have been used reproducibly for decades. Certainly, if a researcher requires an antibody with features that can only be achieved using recombinant methods, then recombinant antibodies may be necessary. Otherwise, there is a wide selection of accepted antibody clones that have been reproducibly manufactured, used and published, meaning it is not imperative to change to recombinant-sourced material solely for that reason.”
Yamamoto elaborates that although recombinant technologies provide an alternative approach to generate monoclonal antibodies, they should be viewed as complementary to using traditional hybridomas rather than as a “cure-all” process for issues surrounding antibodies and their functional robustness and reproducibility. “Simply manufacturing antibodies via recombinant methods will not resolve existing problems,” he says. “Antibodies produced via either method undergo the same downstream manufacturing procedures such as purification, buffer exchanges, and conjugation, each of which can yield functional variability, especially if performed in a poorly controlled manner. This affects all antibodies regardless of how they’re originally sourced. Ultimately, antibody quality must be vetted by functional assessment to confirm every batch performs similarly. The onus is on manufacturers to establish strict, streamlined processes and quality control systems to ensure this for all antibodies, recombinant or not.”
It’s all about choice
The popularity of recombinant antibodies is demonstrated by a growing number of peer-reviewed publications citing their use, and it is clear that they offer many advantages. Not least of these is their suitability to support extended flow cytometry studies. “The long-term reproducibility of recombinant antibodies enables researchers to have confidence that an assay will remain consistent over the lifetime of a given study’s execution, rather than falling victim to unreliable antibody performance that necessitates experiments to be repeated,” notes Austin.
“The overall design of a flow cytometry experiment completely depends on what you are trying to achieve,” says Fiebig. “The interest we’re seeing in species-switched and Fc Silent antibodies is testament to their usefulness in designing new experiments as well as improving already well-established methods, but the real power of recombinant antibody technology is that it provides researchers with so much more choice. We now have all the tools available to cater to even fairly extravagant experimental designs.”