Recombinant Antibodies in Flow Cytometry

Recombinant antibodies have become indispensable tools for flow cytometry due to the advantages that they offer over traditional polyclonal and monoclonal antibodies. We spoke with Mathilde de Jong, Ph.D., Product Manager for Flow Antibodies at Miltenyi Biotec, and Danielle Callahan, Director of Product Management at Proteintech, to learn how recombinant antibodies are benefiting flow cytometry-based research and get some tips for antibody selection.

What are Recombinant Antibodies?

Recombinant antibodies are produced from a known genetic sequence, which can be obtained in several different ways. One approach involves PCR amplification of the antibody variable regions (VH and VL) from an existing hybridoma cell line with species-specific degenerate primer sets, followed by low-throughput and partial-length Sanger sequencing (1). Another strategy is to use antibody phage display, which we have covered in a previous article. The antibody genes are then cloned into an expression vector and transfected into an appropriate host cell line (usually a mammalian cell line such as CHO or HEK293) for in vitro production. Ultimately, knowing the genetic sequence affords tighter control over the antibody that is expressed, which can significantly improve data quality for scientific research.

Benefits of Recombinant Antibodies in Flow Cytometry

While traditional polyclonal and monoclonal antibodies are still widely used in flow cytometry, their limitations have been well documented. Briefly, polyclonal antibodies can exhibit batch-to-batch variability, due to being produced in different animals at different times, and have a high chance of cross-reactivity, due to recognizing multiple epitopes. Hybridoma-derived monoclonal antibodies are often contaminated with antibody impurities, such as additional heavy or light chains, which can impair affinity and specificity (2). To improve experimental reproducibility, pressure is mounting for all antibodies used in research to be sequence-defined and produced recombinantly (3). By choosing recombinant antibodies for flow cytometry, researchers can reap the following benefits:

Superior Batch-to-Batch Consistency

A main advantage of recombinant antibodies is their superior batch-to-batch consistency compared to traditional antibody reagents. “Because recombinant antibodies are highly defined and genetically engineered they are exceptionally stable and consistent in not only their production, but their performance,” explains Callahan. “This equates to more reproducible results in flow cytometry, which can save time, resources, and sample material.”

High Specificity

Recombinant antibodies have high specificity, which minimizes background and reduces cross-reactivity. This property of recombinant antibodies may come from the original hybridoma clone, which will have been carefully selected during development. It may also be introduced through phage display, such as through switching antigens between panning iterations to remove cross-reactive antibodies (4). Another application of panning is to increase the affinity of an antibody for its target, either by using more stringent washing conditions or reducing the amount of immobilized antigen.

To learn more about how recombinant antibodies can reduce background signal in flow cytometry, check out our previous newsletter.

Capacity for Engineering

Knowing the antibody sequence provides a wealth of opportunities for engineering. “The genetic sequence can be modified to tailor the antibody’s properties, such as changing the isotype, optimizing the binding affinity, or improving the recognition of specific antigens,” reports de Jong. “In addition, recombinant antibodies can be ‘humanized’ by replacing non-human antibody regions with human equivalents, which is especially useful In flow cytometry to minimize non-specific binding and background noise.” Engineering also allows for adding epitope tags to antibodies, which can facilitate detection and isolation.

Better Targeting of Challenging Antigens

Phage display has been widely used to generate antibodies against challenging targets. Examples include snake venom toxins, which would be lethal if injected into a mammalian host, and small antigens such as low molecular weight peptides and drug compounds, which often have low immunogenicity. Phage display technology has also been applied for developing antibodies against membrane proteins, which are notoriously difficult to present in their native conformation (5). Notably, because phage display uses just a fraction of the antigen required for animal-based antibody development, it can often represent a more cost-effective option.

Faster Production

While hybridoma technology takes approximately 4-6 months to produce a monoclonal antibody, phage display takes just a few weeks. “Recombinant technology also makes it much easier to scale up production when large amounts of antibody are needed, such as when running a longitudinal flow cytometry study,” says Callahan.

Reduced Animal Use

Another benefit of recombinant antibody production is that it significantly reduces animal use for scientific research. “Recombinant antibody production aligns with the Replace-Reduce-Refine (3R) principle, giving it an ethical advantage over traditional methods,” notes de Jong. With organizations such as the EU Reference Laboratory for Alternatives to Animal Testing advocating a shift toward non-animal-derived antibodies, recombinant technology has a pivotal role in shaping the future of antibody-based research.

Available Options

Both Miltenyi and Proteintech offer a comprehensive selection of recombinant antibodies for flow cytometry-based research, with available options including the following:

REAfinity™ Recombinant Antibodies

Miltenyi’s REAfinity Recombinant Antibodies are optimized for flow cytometry and incorporate innovative design features to simplify experimental planning and ensure more reliable results. “All REAfinity antibodies share the same human IgG1 isotype, eliminating the need for multiple isotype controls,” explains de Jong. “REAfinity antibodies also contain a mutated human IgG1 Fc region, which prevents binding to Fc receptors on effector cells and thus gives background-free analysis.”

To learn how Miltenyi validates REAfinity antibodies for flow cytometry, visit its Recombinant Antibodies for Flow Cytometry Applications page. And, for examples of how REAfinity antibodies are being used, publications are available that describe the monitoring of human immunodeficiency virus (HIV)-infected patients, the genetic engineering of NK cells for CAR-enhanced immunotherapy, and the development of neoantigens targeting metastatic breast cancer, among many other applications (6,7,8).

Uni-rAb™ Recombinant Monoclonal Antibodies

Proteintech’s Uni-rAb Recombinant Monoclonal Antibodies are developed with the company’s Antigen-Specific B-Cell Cloning & Engineering (ABCE™) platform, which uses multi-parametric FACS to isolate antigen-specific B cells from immunized animals. “An advantage of the ABCE platform is that it maintains native VH-VL pairings,” reports Callahan. “This is critical for antibody specificity and functionality, yet is often lost in approaches such as phage display. Additionally, because the ABCE platform is based on single B-cell screening, it yields more positive clones in less time compared to traditional hybridoma-based approaches.”

Besides its Uni-Rab products, Proteintech also offers a growing selection of recombinant nanobodies for flow cytometry. “Recombinant nanobodies provide similar advantages to recombinant IgG antibodies, but with the added benefit of their small size, which lets them bind more easily to epitopes in crowded cellular environments,” says Callahan. All of Proteintech’s recombinant antibodies and nanobodies for flow cytometry can be found here.

Selecting Recombinant Antibodies for Flow Cytometry

Choosing recombinant antibodies for flow cytometry is no different to selecting traditional antibodies. “A good starting point is to check that the antibody is validated for flow cytometry and suitable for your sample type and species,” advises Callahan. “This should include reviewing the product datasheet for relevant performance data.”

It is also important to confirm whether the target antigen is expressed on the cell surface or intracellularly, as this will help to determine your staining protocol. “If you are unsure which experimental set-up to choose, check out Miltenyi’s step-by-step application protocols and flow cytometry antibody panels,” suggests de Jong.

Another factor to consider is the choice of antibody conjugate. “Your fluorophore should provide good signal intensity without being so bright it causes spillover into adjacent channels and compensation issues,” says Callahan. “Often, bright dyes are best paired with low abundance targets and dim dyes with more abundant proteins.” The fluorophore should also be compatible with your flow cytometer’s laser and filter settings – if you’re unsure, you can use FluoroFinder’s Flow Cytometry Panel Design tool.

For flow cytometry applications where Fc receptor binding might be problematic, such as when working with samples that contain FcγR-expressing cells, you may want to think about using engineered antibodies. Here, REAfinity antibodies or isotype-switched antibodies can help to reduce unwanted background signal from non-specific binding.

Testing your chosen antibody with appropriate controls is critical for verifying its correct performance. De Jong recommends using an isotype control antibody to assess non-specific binding and background staining and Fluorescence Minus One (FMO) controls to accurately set gating thresholds and compensate for spillover between channels.

Finally, be sure to purchase your antibody from a trustworthy vendor. Your chosen partner should be able to answer questions about the antibody and offer technical support where needed.

Supporting Your Research

Whether you’re using traditional or recombinant antibodies for flow cytometry, FluoroFinder has all the tools you need to streamline experiment design. Use our Antibody Search function to find antibodies that are validated for flow cytometry, then check out our Spectra Viewer to compare the spectral properties of more than 1,000 dyes alongside instrument-specific laser and filter configurations.