Antibodies 101: Fab Fragments

Imagine an antibody. Do you immediately visualize a Y-shaped protein reminiscent of the Addgene mascot Abi? If so, you are not alone. Full-sized antibodies dominate the world of research affinity reagents, and for good reason. However, sometimes you want a tool that is a little more compact — more of an antibody fragment rather than the whole thing. Fab fragments are exactly that! Here, we’ll go over what a Fab fragment is and when you might use it in the lab.

What is a Fab Fragment?

Consider the classic IgG antibody (Figure 1): it is composed of four peptide chains (two identical heavy chains and two identical light chains) each with a constant region and a variable region. The chains are covalently linked to one another by disulfide bonds between the two heavy chains and between one heavy chain and one light chain. When assembled, the four chains make a Y-shaped protein that can be divided into the Fragment Crystallizable region (or the “Fc”) and two Fragment Antigen Binding regions (or “Fabs”). The Fc region is the “tail” of the antibody and comprises portions of the constant regions of the two heavy chains. In the lab, this is the region that determines what secondary antibody you should use when performing indirect immunoassays. The Fab regions, on the other hand, are the “arms” of the Y and comprise the entire light chain and a variable and constant region of the heavy chain. As the name implies, these Fabs are the regions of the antibody that actually bind antigens. 

Figure 1: IgG antibody structure with important features labeled. V = Variable domain. L = Light chain. C = Constant domain. H = Heavy chain. Created with biorender.com  

The modularity of antibodies means that it is relatively easy to physically divide up the different regions, both through genetic and proteolytic approaches. This comes in handy when, for example, you want to swap out one isotype for another. But it also allows you to use just a fragment of the antibody in isolation from another portion. For example, Fab fragments are molecules composed of just the Fab portion of the antibody. Fab fragments are useful when you just need an antibody’s antigen binding function but not the Fc region (more on when that might be later).

Find scFv plasmids at Addgene

Depending on how an antibody is broken up, there are a few commonly available different fragments that retain antigen binding (Figure 2): 

• F(ab')2 - These fragments are produced by proteolytic digestion that retains an antibody’s disulfide bonds within the hinge region. The resulting product is a mini-Y with both Fab regions still attached to one another. Size: ~110 kDa.
  
  
• Fab' - These fragments are produced by mild reduction of F(ab')2 fragments, which reduces the hinge region disulfide bonds, resulting in two independent Fab fragments with small tails of the hinge region. Size: ~55 kDa. 
  
  
• Fab - These fragments are produced by proteolytic digestion above the hinge-region, which results in two independent Fab fragments. Size: ~50 kDa.
  
  
• Fv - These fragments are composed of just the variable domains of the heavy and light chains of an antibody. Arguably not a “Fab” fragment, Fvs are often produced recombinantly and engineered to be joined by a linker peptide, resulting in a product called a single-chain Fragment Variable (scFv). Size: ~25 kDa.

Figure 2: Comparison between a full IgG antibody and common antigen binding fragments. Created with biorender.com.

Why Fabs? 

Antibody fragments have a handful of advantages over full antibodies. Their smaller size allows them to get into samples more efficiently and can also improve localization accuracy when used in super-resolution microscopy by reducing the distance between the target and reporter. They have fewer non-specific binding opportunities due to the lack of an Fc region. The lack of Fc also simplifies analyses when used in structural studies and reduces immunogenicity when used in vivo.    

Of course, the lack of an Fc portion means that most of the common anti-IgG secondary antibodies you already have in your refrigerator will not recognize a Fab fragment. In order to detect a Fab fragment, it either needs to be directly conjugated to a reporter or you need a light-chain specific conjugated secondary antibody. These requirements limit the flexibility of and demand for Fab fragments and subsequently Fab fragments intended for use as primary antibodies are relatively uncommon. Though, if you want to use Fab fragments as primary antibodies, you can make your own antibody fragments as needed. 

In contrast, anti-IgG antibodies, commonly used as secondary antibodies, have much broader applicability. It is fairly easy to find these in a Fab fragment format. Reporter-conjugated anti-IgG Fab fragments make good secondaries for cases where you need to pre-form primary-secondary antibody complexes before applying them to samples, such as when performing IHC in a sample where your secondary antibody recognizes endogenous antigens. In these cases, even though one full antibody may be able to get into the tissue, two full antibodies bound together may be too large. Using a complex that is one full antibody bound to a Fab fragment gives you a better chance of getting the complex into the sample. Alternatively, Fab fragments can be used as a block in similar scenarios. By applying unconjugated Fab fragments that are the same species as your secondary antibody to your sample prior to adding your primary and secondary antibodies, you block the antigens that the secondary would recognize and reduce non-specific binding.  

At this point, you may be asking yourself, what type of Fab fragment are we talking about here? Many of the Fab fragments described above can be used for similar applications and have similar advantages, though with some caveats. 

First off, F(ab’)2 fragments are bivalent, so may have higher avidity than monovalent Fab’ or Fab fragments. Higher avidity means that F(ab’)2 fragments may have overall stronger interaction with their antigens than a Fab or Fab’ fragment. But this bivalency may cause problems for blocking purposes, since the fragment could bind not only the endogenous proteins you are trying to block but your primary antibody as well. 

Next, Fab and Fab’ fragments are almost interchangeable, but the extra bit of heavy chain on a Fab’ can act as a convenient site for conjugation. 

Finally, Fvs are the most amenable to recombinant expression and will have the highest tissue penetration. However, they tend to be less stable and have lower binding affinity than some of the larger fragments. 

Think of antibody fragments as new components in your antibody tool box. As you consider which tools to use, be sure to think about what pieces of the antibody you do and don’t need, and how the protein will interact with other components of your experiment. These considerations will help you narrow down which antibody format to use for your specific scenario.   

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References and resources

References

Bates A, Power CA (2019) David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments. Antibodies 8(2):28. https://doi.org/10.3390/antib8020028   

Wessel GM, McClay DR (1986) Two embryonic, tissue-specific molecules identified by a double-label immunofluorescence technique for monoclonal antibodies.  J Histochem Cytochem : Off J Histochem Soc 34(6):703–706. https://doi.org/10.1177/34.6.3084626 

Zhang Q, Miyamoto A, Watanabe S, Arimori T, Sakai M, Tomisaki M, Kiuchi T, Takagi J, Watanabe N (2022) Engineered fast-dissociating antibody fragments for multiplexed super-resolution microscopy. Cell Rep Methods 2(10):100301. https://doi.org/10.1016/j.crmeth.2022.100301 

Additional resources on the Addgene blog:

Introduction to Antibodies

Single Chain Fragment Variables (scFvs)

Avoiding the Mouse on Mouse Mess in IHC

Resources on Addgene.org 

Browse ready-to-use antibodies at Addgene