Labeling and purifying antibodies in-house can be a multi-step process. The alternative—purchasing prelabeled antibodies from a supplier—is typically less labor- and resource-intensive, but limits the user’s ability to select the best-performing antibodies and dyes. New technology removes the traditional column purification step required with in-house labeling while maintaining the quality of the final reagent.
In-house fluorescent-antibody labeling
Fluorescent labels are chemically attached to antibodies to aid in the detection and quantification of cellular proteins, often referred to as targets or antigens. These may be located on the cell surface, expressed internally or secreted into the extracellular environment. Labeled antibodies are essential in many protein analysis techniques, such as flow cytometry, immunohistochemistry, fluorescent cell imaging and Western blotting.
An alternative to using prelabeled antibodies is to perform labeling reactions in-house. Dyes, enzymes and other molecules can be linked to antibodies using well-defined chemistries. Direct amine-succinimidyl ester conjugation is a good choice for fluorescent labels. Two-step reaction methods are also widely used, and include thiol–maleimide coupling (also for fluorescent labels), amine–carboxyl coupling (for magnetic bead and chip surfaces) and aldehyde–hydrazide coupling (for horseradish peroxidase). Of these, fluorescent labeling with succinimidyl esters is the most common because it provides a high label-to-protein ratio that enables sensitive detection of antigens by flow cytometry and cell imaging.
In the first step of antibody labeling, a fluorescent dye with an amine-reactive succinimidyl ester is added to the solution containing the antibody. The reaction progresses quickly, forming covalent bonds that are basically the same as natural peptide bonds. These bonds prevent dissociation of the fluorophore from the antibody. In this reaction, the antibody is the limiting reagent, which means that excess free dye is present in the sample. Free dye is typically removed from the final preparation using column chromatography.
Column chromatography can isolate a single molecular species from a complex mixture. However, running a purification column after labeling an antibody is not the preferred method. In fact, column purification was once the only way to prepare fluorescent antibodies. Even with newer spin-column methods that reduce the time and complexity of antibody cleanup, batch-to-batch variation and loss of reaction yield occur.
Column-free fluorescent labeling
Column-free methods for antibody labeling replace the purification step. Quench buffer, or quencher, added to the final mixture (see Figure 1) binds to the free-dye molecules in solution, switching off their inherent fluorescence. No purification is needed, and the tagged antibody can be used immediately in immunostaining experiments without removing the quenched dye.
Figure 1 – Antibody labeling kits with quenchers eliminate the purification step for in-house labeling.ReadiLink antibody labeling kits from Bio-Rad Laboratories (Hercules, Calif.) are one method that eliminates the column purification step. Antibodies labeled in this manner are suitable for the same applications as traditionally labeled antibodies, including flow cytometry and cell imaging. Available in 13 different excitation and emission wavelength combinations, the kits can accommodate multi-color experiments across a range of instrument configurations. In a recent study,1 the ReadiLink kit was used to directly label a monoclonal antibody against T-cells and neutrophils. This conjugate allowed researchers to visualize immune cell infiltration into wound sites by fluorescence microscopy.
Prelabeled antibodies: pros and cons
Prelabeled antibodies against targets such as CTLA4 (CD152) and PD-1 (CD279), which are used in immuno-oncology research, can be the most practical choice in many situations. Knowing which antibodies work ahead of time makes purchasing prelabeled antibodies a low-risk option for those who want to quickly publish their work.
For researchers who work with new targets and antibodies, however, there are substantial trade-offs with prelabeled antibodies, one being the limited number of ready-made fluorescent antibodies from which to choose. Prelabeled antibodies reduce the ability to customize an experiment, which means that scientists will need to do their own labeling. When designing a multi-color assay panel for flow cytometry, each antibody must not only be specific for its intended antigen, but also have an excitation/emission profile that is compatible with other fluorophores in the panel, as well as the cytometer’s laser, detector and filter set configuration. The flexibility to fill in gaps with a custom fluorophore/antibody combination could be the key to a successful experiment.
Another drawback is that it can be difficult to manage expensive reagent inventories. Because fluorescent conjugates have a shorter shelf life than unlabeled antibody, it is not advantageous for researchers to commit to quantities of labeled product. Labeling just 10 or 20 μg of test antibody at a time can save money. Additionally, researchers might purchase a labeled secondary antibody, only to find that they need to label the primary antibody for increased specificity.
Antibody specificity and sensitivity
Further complicating the use of prelabeled antibodies is the process of trial and error that goes into determining which antibodies are sensitive and specific for binding an antigen. Off-the-shelf reagents can be of poor quality or may have undergone minimal testing in the application of interest. Yet antibody specificity is critical to running a valid experiment. Indeed, use of a nonspecific antibody can lead to erroneous and irreproducible results.2,3 Some experts argue that validation is necessary with each new antibody and lot.4–7 Prelabeled antibodies limit the ability to optimize antibody selection.
Summary
Antibody labeling is routine for life science researchers working in disciplines from basic cell biology to disease research. Prelabeled antibodies are the best option in many cases. In others, column-free methods provide the flexibility to use the optimal combination of antibodies and dyes.
References
- Qian, L.-W.; Leung, K.P. et al. Wound Rep. Reg. 2015, 24, 26–34.
- Baker, M. Reproducibility crisis: blame it on the antibodies. Nature 2015, 521, 274–6.
- Egelhofer, T.A.; Klugman, M.A. et al. An assessment of histone-modification antibody quality. Nat. Struct. Mol. Biol. 2011, 1, 91–3.
- Begley, C.G.; Buchan, A.M. et al. Robust research: institutions must do their part for reproducibility. Nature 2015, 525, 25–7.
- Begley, C.G. and Ellis, L.M. Drug development: raise standards for preclinical cancer research. Nature 2012, 483, 531–3.
- Acharya, P. How can antibodies ensure your research stands the test of time? Biosci. Technol. 2016; http://www.biosciencetechnology.com/articles/2016/01/how-can-antibodies-ensure-your-research-stands-test-time.
- Bogoev, R. The antibody challenge: Bio- Rad’s precise solution. BioRadiations 2015.
Chris Linnevers is global product manager in the Cell Biology Division of Bio-Rad Laboratories, 2000 Alfred Nobel D., Hercules, Calif. 94547; tel.: 510-741-5876; e-mail: [email protected]; www.bio-rad.com