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What are the 2 methods for labelling antibodies?

Maarten Brom, PhD

There are various types in antibody labeling, including conjugating antibodies to fluorophores, radioisotopes, and nanoparticles.
Can antibodies be labeled with fluorescent molecules? Yes, there are many options to choose from. In this article, we discuss the 2 methods of labelling antibodies. We first focus on antibody fluorescent labeling and the 2 main methods. What are the 2 methods for labelling antibodies?

Direct and indirect labeling of antibodies

The first method is direct labeling, where the label is directly conjugated with your antibody. The second method is indirect labeling, where the label is conjugated with a secondary antibody that then is coupled with the primary antibody. Good to know already, both methods for labeling antibodies are used in fluorescence-based imaging applications and can be used in drug development.

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Difference between direct and indirect antibody labeling

You now know that the 2 methods for labeling antibodies are direct and indirect. A big difference between the two is that direct labeling can be used for in-vivo imaging while indirect labeling is mostly used for in vitro. For example enzyme-linked immunosorbent assay (ELISA) and fluorescence microscopy. Which component of a fluorescent antibody test is fluorescently labeled? In all cases, it is the fluorophore that emits the fluorescent signal upon excitation with light of a specific wavelength. In direct labeling, the fluorophore is directly conjugated with the antibody, while in indirect labeling this is done with a secondary antibody. We’ll cover the advantages and disadvantages of each method.

Direct antibody labeling

Direct antibody labeling is a simple method where the label is coupled to the antibody. With direct labeling fluorophore conjugated antibodies don’t require an additional antibody.

Benefits of direct labeling antibody

  1. Quick antibody labeling protocol with fewer additional steps
  2. Fewer chances of experimental error
  3. Reduced risk of label detachment
  4. High labeling efficiency where most of the antibody molecules are successfully labeled
  5. Less interference with the binding properties of the antibody
  6. Usable for research, diagnosis, and image-guided surgery

Indirect antibody labeling

Indirect antibody labeling is the second of the 2 common methods for antibody labeling. In this method the label is not attached to the primary antibody but to a secondary antibody. There can be even more than one ancillary antibody. What are the advantages of indirect labeling in comparison to direct antibody labeling?

  1. Because the secondary antibody can be used as an amplifier, indirect labeling is more sensitive and versatile
  2. The same secondary antibody can be used on multiple primary antibodies
  3. No need of labeling your primary (targeting antibody)
  4. Large availability of commercially available fluorescent labeled antibodies to detect your primary antibody
  5. Possibility of multiplexing where secondary antibodies with different labels can be used to detect multiple primary antibodies

How to determine which method of antibody labeling to use

The choice between direct and indirect labeling of antibodies is made on the type of experiment that you want to perform. For in vivo (and ex vivo) imaging, direct labeling would be the labeling method of choice. For in vitro experiments you can choose for indirect or direct labeling. When it comes to specificity and sensitivity, indirect antibody labeling is often the preferred method. Indirect labeling is, in general, easier since a large selection of fluorescent secondary antibodies to detect your primary antibody are commercially available. This means that you don’t need to label your antibody with a fluorescent dye, with the risk of altering the (function of) your antibody. Sometimes, a (proper) secondary labeled antibody is not available and you need to label your targeting antibody directly.

Fluorophores for labeling antibodies

Many fluorophores, also referred to as fluorochromes or fluorescent dyes, can be used and even multicolor experiments can be made. This allows multiple targets to be studied with for instance flow cytometry and epifluorescence microscopy by using fluorescent tagged antibodies. A fluorochrome conjugated antibody can be used for in vivo, ex vivo and in vitro (ELISA, flow cytometry, and fluorescence microscopy) experiments.

What is fluorescent labeling of antibodies?

Fluorescent labeling of antibodies means coupling the antibody with a fluorescent dye, resulting in a fluorochrome conjugated antibody. The fluorescent dye is coupled to the antibody using a functional group (for example NHS-ester or maleimide). The type of dye depends on the application. The wavelength of the emitted light has a strong influence on the detection of the signal in various applications. High wavelengths are more suited for in vivo imaging, as the penetration depth is higher and the background signal is lower

What are commonly used fluorophores?

Fluorescein isothiocyanate (FITC), Alexa fluor, and Cyanine dyes are commonly used fluorophores and are directly conjugated to the antibody. As you can see many of them can be used in both methods. In general, fluorescent dyes with light emitted at a higher wavelength are used for in vivo and ex vivo imaging. These dyes have a higher penetration depth and lower background signal.

Common fluorophores:

  • Cy dyes
  • Cy3
  • Cy3.5
  • Cy5
  • Cy5.5
  • Cy7
Other fluorophores

  • Fluorescein isothiocyanate (FITC)
  • Rhodamine B
  • Alexa fluorophore
  • Alexa Fluor 488
  • Alexa Fluor 594
  • Alexa Fluor 647

Creating a labeling strategy

With all the choices to be made, it’s clear you need a labeling strategy. We’ve discussed direct and indirect labeling, but there is more to consider. Both labeling methods can be performed in several ways, for instance, genetic engineering, chemical modification, and enzymatic conjugation (mostly used in indirect labeling). All the above can be used in site-specific antibody labeling to increase accuracy and specificity. At TRACER we specialize in labeling compounds, like antibodies. We can help you with your labeling strategy. We are a CRO, Contract Research Organization, helping drug developers with the fastest and most effective clinical trials by using molecular imaging.

More than just fluorophores

It’s true that using fluorophores is the most common imaging method when labeling antibodies. But radioisotopes for radiolabeling can also be considered. Zirconium 89 for instance can be used for radio immuno PET imaging of a large variety of tumors. Your antibody can be coupled with a radiolabel that emits radiation which can be used for imaging. T

Read about finding the optimal tracer for your compound, or contact us for tailored advice on your project.

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