Antibody Labeling Principles, Advantages, and Considerations

Labeled antibodies are essential tools for many different research applications, spanning single-analyte western blots to cytometry by time-of-flight (CyTOF) experiments capable of detecting 50 or more targets. But if the specific antibody conjugate you need is not available commercially, you may have to perform antibody labeling in-house. Here, we look at the basic principles of antibody labeling and offer some practical guidance.

Antibody labeling principles

A main reason for using labeled antibodies is to enable target detection with various immunoassay techniques. Common labels include enzymes, fluorophores, and biotin, as well as colored latex beads, gold nanoparticles, and metal tags, all of which are typically conjugated to antibodies using one of two labeling chemistries. The best-known approach targets the antibody’s amine (NH₂) groups, which are found in lysine residues and at the N-termini of polypeptide chains and allow for relatively easy labeling at multiple sites. Alternatively, antibodies may be labeled at sulfhydryl (SH) bearing cysteines, which are predominantly clustered at the hinge region. While secondary antibodies are almost always labeled, it is increasingly common for primary antibodies to be labeled in a similar manner.

Advantages of using labeled primary antibodies

Labeled primary antibodies can offer several advantages for scientific research. According to Danielle Callahan, Director of Product Management at Proteintech, these include more streamlined workflows and simpler multiplexing. “By removing the additional protocol steps required by indirect staining, labeled primary antibodies make experimental workflows shorter and less complex,” she says. “It is also possible to combine several labeled primary antibodies from the same host species for multiplexed experiments without worrying about possible secondary antibody cross-reactivities that could lead to false-positive results.” A further benefit of using labeled primary antibodies is that potential background signal resulting from secondary antibody binding to endogenous immunoglobulins or other sample components is eliminated.

FFPE human kidney was stained with rabbit antibodies to Calbindin (labeled with FlexAble CoraLite^® Plus 555, orange), ACE2 (labeled with FlexAble CoraLite^® Plus 647, magenta), and Podocalyxin (labeled with FlexAble CoraLite^® Plus 488, green). Nuclei are stained with DAPI (blue). Image provided by Proteintech.

Reasons to label antibodies in-house

Although a growing number of labeled antibodies can be purchased as off-the-shelf products, researchers may still need to perform labeling in-house. “Maybe you have developed an antibody yourself and so that antibody is not available commercially,” suggests Emily Cartwright, Ph.D., Senior Product Marketing Specialist at Bio-Techne. “Or perhaps you have an unlabeled antibody that is validated in a particular application, but you wish to test whether a conjugated version of that same antibody will work in a different application.” Performing labeling in-house can also help to increase flexibility when designing multiplexed panels by letting researchers choose which fluorophores or other molecules to pair with each antibody. “For example, if an antibody is used for identifying a protein of low abundance, you may decide to label it with a brighter fluorescent dye,” explains Callahan.

Multiplex assays are driving demand

The greater use and sophistication of multiplex assays has led to the development of countless antibody conjugates. “Through our Novus Biologicals brand, Bio-Techne currently offers more than 20,000 conjugated antibodies backed by our quality guarantee,” says Cartwright. “This includes antibodies conjugated to over 25 fluorescent and enzymatic labels, so if you’re looking for a labeled commercially available antibody, there is a high probability that we already have it. In situations where that is not the case, you can easily label your own antibodies with our ready-to-use conjugation kits.”

Similarly, for CyTOF, the number of ready-to-use labeled antibodies continues to rise. “Antibodies to over 400 unique human or mouse targets, pre-labeled to different tags, are available through Standard BioTools™,” reports Leslie Fung, Flow Cytometry Product Manager at Standard BioTools. “These can be added into pre-built panels to streamline scientific research. For example, our Maxpar® Direct™ Immune Profiling Assay™ is a core panel for detecting 30 markers, which can be easily augmented with defined expansion panels to cover cell functions including activation, exhaustion, and cytokine secretion. Panels can also be customized using catalog antibodies to incorporate targets of interest or, if a desired antibody is not yet available, we offer Maxpar^® Labeling Kits for tagging IgG antibodies with a choice of up to 42 different metals.”

Factors to consider for antibody labeling

If you do find yourself having to label antibodies in-house, there are several important factors to consider. “Critically, you will want to think about how much antibody is required for the labeling reaction and how that antibody is formulated,” cautions Callahan. “Most conjugation methods require 100 ug or more of antibody in a PBS only buffer at a certain concentration, which can limit your options. In contrast, Proteintech’s FlexAble Antibody Labeling Kits let you label as little as 0.5 ug of antibody without any of these restrictions, meaning you can prepare the amount of antibody you need for your experiment and save the unconjugated antibody for other applications or labeling with additional moieties. FlexAble is currently offered in six different fluorophore formats and more detection types will be introduced soon.”

Labeling IgM antibodies

During the past few decades, IgM antibodies have seen increased interest for immunostaining applications. “IgM is a pentamer with many more binding sites than IgG, which can provide greater avidity for target proteins,” explains Eric Torres, Ph.D., Marketing Manager at Biotium. “However, with very few conjugated IgM antibodies available and a longstanding belief that IgM is difficult to label because it is unstable at the basic pH used for conjugation reactions, researchers have struggled to capitalize on this benefit. To make IgM antibodies more accessible to researchers, Biotium recently launched our Mix-n-Stain™ CF^® Dye IgM Antibody Labeling Kits. These provide optimal labeling of IgM antibodies to produce bright high-performance conjugates without the need for downstream purification and are available with eight different CF^® Dye colors to allow for flexible panel design.”

Future perspectives

With labeled antibodies being such valuable tools for scientific research, the range of products on offer looks set to evolve. “Commercial availability of antibody conjugates and antibody labeling kits has significantly expanded what can be interrogated and how quickly,” comments Fung. “If you need to label your own antibodies, you will find comfort in knowing that conjugation can be straightforward and there is often no special equipment or experience required.”