Putting secondary antibodies first

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Our bodies have the capability to fight cancer. Most of the time cancer cells are immediately recognized and eliminated via sentinel cells that make up our

body’s immune system. Sometimes, though, cancer cells go undetected. Immuno-oncology uses therapies and tools to boost the immune system’s ability to recognize and respond to these rogue cells. Immunohistochemistry (IHC) and flow cytometry are just

two of the many techniques used in immuno-oncology. The most important element of these techniques are two sets of antibodies: primary antibodies that bind to target molecules such as cancer markers, followed by reporter-labeled secondary antibodies that enable identification using imaging or detection techniques. While primary antibodies usually get the most attention from researchers, secondary antibodies are often crucial for the success of an experiment. In supporting immuno-oncology, Jackson

ImmunoResearch provides a wide range of secondary antibodies and their respective reporter conjugates. The increasing complexity of immuno-oncology experiments and the expanding development of treatments drive the need for even more commercially available reagents that scientists can trust.Early detection is paramount in cancer research,

diagnosis, and therapy, but the targets and required sensitivities remain elusive. Instead of just looking for tumors or abnormal blood counts, scientists and clinicians study gene expression, cell markers, and other molecular indicators of cancer. In many cases, they can see more

by using a secondary antibody to amplify signal from a primary antibody directed against a cancer-related target. Secondary antibodies are essential for revealing targets with little or no background, including in applications such as flow cytometry.“Secondary antibody conjugates can improve a

flow cytometry experiment by providing options if no conjugated primary [antibody] is available, or where the active site of the primary antibody needs to be preserved, and by signal amplification, if the antigen is poorly expressed,” says Diane Sharp, supervisor of Product Development at Jackson ImmunoResearch. At the Côte d’Azur University in Nice, France, professor

of pathology Paul Hofman says that using secondary antibodies in IHC “is a daily practice in our lab.” As an example, he points out that he uses secondary antibodies in analyzing lung cancer samples for programmed death-ligand 1 (PD-L1), which can block immune cells from identifying and killing the cancer cells. Forms of immunotherapy directed at blocking PD-L1 allow a patient ’s immune cells to fight the lung cancer. Hofman and his colleagues test both biopsy and surgery samples for this protein using primary antibodies, such as 22C3, SP142, and SP263, which can be located with a secondary antibody. By using imaging to identify the secondary antibodies,

Hofman can also achieve multiplexing. “We can do comparisons to different monoclonal antibodies at the same time,” he says. In fact, thanks to highly specific secondary antibodies, he and his colleagues have IM

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developed IHC-based tests that simultaneously analyze PD-L1 and other cancer-related cell markers, such as CD8, TTF1, and P40, as well as predictive biomarkers for targeted therapy, such as ALK, BRAF, and ROS1.

Adding optionsHowever, just looking at tissue

sections cannot necessarily provide the earliest indication of cancer or the impact of treatment. With a liquid biopsy, for example, clinicians can analyze a blood sample for indicators of cancer, such as circulating tumor cells (CTCs). “We’ve done lab research on PD-L1 in CTCs,” Hofman says.Other scientists also note the need

to monitor cancer with antibodies. Timothée Chanier and Patrick Chames of Aix-Marseille University in France wrote of immunotherapies that “it is becoming increasingly important to monitor and modulate the tumor immune infiltrate for efficient diagnosis and therapy” (1). They added that so-called single-domain or VHH antibodies—also called Nanobodies, which are recombinantly produced and small due to the absence of a light chain—penetrate tissue more easily than ordinary antibodies.Jackson ImmunoResearch offers a wide range of anti-

VHH secondary antibodies that are useful in immuno-oncology. For example, chimeric antigen receptor (CAR) T cell therapy has had more success in treating hematological cancers than it has in treating solid tumors. The main reason for this difference is the limited number of tumor-specific targets that can be recognized by CAR T cells. Also, traditional antibodies used by CAR T cells trigger an attack by the body’s other immune cells before those cells can bind to the tumor. However, due to their small size and high affinity to target antigens, VHH antibodies are able to better detect and reach tumors before being taken out by the immune system. Now VHH-based conjugates, such as nanobody–enzyme, nanobody–exotoxin, and nanobody–T cells are becoming potential strategies in tumor-targeting treatments (2,3). Characterization of these modified T cells is typically performed by flow cytometry using anti-VHH secondary antibodies.

Relying on reagentsScientists are able to accelerate

their research and develop IHC-based cancer tests because they have convenient access to specific primary and secondary antibodies. “All of the reagents that we use are commercially available,” Hofman says. “We never develop any ourselves.” Scientists use commercially available reagents in part because it ’s easier—they can simply order them. Manufacturing standards ensure product quality, which leads to more consistent results. In addition, commercially available reagents help in other ways. For example, scientists in Hofman’s lab work on in vitro diagnostic (IVD) tests, and he says, “we use commercially available reagents for our IVD projects, because that makes it easier to get CE [Continuing Education] accreditation.”Other experts agree on the benefits

of commercial reagents. As director of the University of Washington’s histology and imaging core, Charles Frevert uses lots of IHC for image-based analysis. “We validate and optimize all antibodies, and we use high-quality reagents,” he says. “Jackson ImmunoResearch has a

good reputation for that.” Plus, the company’s products are manufactured under ISO 9001:2015 certification.From basic research in immuno-oncology to diagnostics

and treatments, secondary antibodies improve imaging and flow-based work. As the field evolves with tools such as engineered antibodies, scientists and clinicians will continue to need more tools—especially easily obtainable reagents—to make further progress.

References1. T. Chanier, P. Chames, Antibodies 8 , 13 (2019).

2. P. Bannas, J. Hambach, F. Koch-Nolte, Front. Immunol. 8 , 1603 (2017).

3. A. Sircar, K. Sanni, J. Shi, J. J. Gray, J. Immunol. 186 , 6357–6367 (2011).

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Top , Human ileum mucosa stained for type IV collagen (Cy2, recolored red), PGP 9.5 pan-neuronal marker (Cy3, recolored green/aqua), and Ulex europaeus agglutinin (Cy5, recolored blue). Secondary antibodies conjugated with Cy2, Cy3, and Cy5. Bot tom , Epidermal basement membrane labeled with mouse anti-collagen IV + donkey anti-mouse-Cy2 (green), keratinocytes and endothelium labeled with biotinylated Ulex + Streptavidin-Cy5 (blue), and nerve fibers labeled with rabbit anti-PGP9.5 + donkey anti-rabbit-Cy2 (green).

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