サポート ブログ Antibody Basics: Part 5 - Antibody Formats: Antibody fragments (Fab, F(ab')2, Fc)

Antibody Basics: Part 5 - Antibody Formats: Antibody fragments (Fab, F(ab')2, Fc)

Biointron 2024-04-10 Read time: 10 mins

Welcome back to Antibody Basics by Biointron, Part 5. In this episode, we’ll continue to cover antibody formats, specifically, Fab, Fab2, and Fc fragments.

What is antibody fragmentation?

  • Biochemical or genetic cleavage of antibodies into smaller fragments. This process is used in various laboratory and clinical applications, as fragments retain the specific targeting capabilities of full monoclonal antibodies while altering physiochemical features. 

  • There are several different types of fragments available, such as small monovalent antibody fragments (Fab, scFv) and engineered variants (diabodies, triabodies, minibodies and single-domain antibodies). 

What are Fab, Fab’2, and Fc?


Fab (Fragment antigen-binding):

  • Formed by enzymatic cleavage of the antibody molecule with papain, resulting in two identical Fab fragments

  • Contains the variable regions of both the heavy and light chains

  • Each Fab fragment contains one antigen-binding site, and is responsible for binding to antigens


F(ab’)2:

  • Formed by enzymatic cleavage of the antibody molecule, but with pepsin instead of papain

  • F(ab')2 fragments consist of two Fab portions connected by disulfide bonds in the hinge region of the antibody

  • Lacks the Fc region and is bivalent, meaning it can bind to two antigen molecules simultaneously

Fc (Fragment crystallizable):

  • Contains the constant regions of the heavy chains 

  • Involved in various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement activation, and binding to Fc receptors on immune cells

  • Plays a crucial role in the immune response and antibody-mediated clearance of pathogens or target cells.

The emergence and evolution of antibody fragments


1949-1960: Rodney Porter introduces the concept of enzymatic digestion with papain to cleave antibodies into Fab fragments and an Fc region.

1960: Alfred Nisonoff used pepsin on rabbit IgG and observed the generation of a single bivalent fragment F(ab’)2.

1972: Rodney Porter and Gerald Edelman won a Nobel Prize for independently determining the basic structure of immunoglobulins, revealing the presence of two identical heavy chains and two identical light chains.

20th Century: Advances in genetic engineering and recombinant DNA technology enable the production of engineered antibodies with modified Fab, F(ab')2, and Fc regions, leading to the development of therapeutic antibodies.   


2000s-Present: Continued R&D efforts focus on optimizing stability, specificity, and pharmacokinetics, leading to the emergence of novel formats such as bsAbs and ADCs.

Enzymatic digestion with proteases

Papain digestion:


  • Papain is a cysteine protease which specifically cleaves peptide bonds adjacent to amino acids with bulky hydrophobic residues, such as phenylalanine, tyrosine, and tryptophan. 

  • In IgG, papain cleaves the hinge region between the Fab and Fc fragments, resulting in the formation of two identical Fab fragments and an Fc fragment.

Pepsin digestion:


  • Pepsin is an acidic protease that preferentially cleaves peptide bonds at the N-terminal side of aromatic amino acids, particularly phenylalanine and leucine, under acidic conditions. 

  • In IgG, pepsin cleaves near the hinge region but closer to the Fc portion compared to papain digestion, resulting in the generation of F(ab')2 fragments.


Ficin digestion:


  • Ficin is a cysteine protease extracted from the latex of the fig tree and exhibits specificity towards peptide bonds adjacent to amino acids with bulky hydrophobic residues, similar to papain.

  • In IgG, ficin can generate either Fab or F(ab')2 fragments, depending on the concentration of cysteine activator used.

Diagnostic applications

Use as secondary antibodies in immunoassays. Fab fragments are useful in double staining experiments to block background signals from primary antibody binding. F(ab’)2 fragments are useful to avoid nonspecific binding between Fc receptors on cells or to Protein A/G. Due to having two binding sites, they can also both bind and precipitate antigens.

  • Immunohistochemistry: Visualizes protein distribution in tissue sections using antibodies tagged with enzymes or fluorescent dyes.

  • Immunofluorescence: Identifies protein localization through fluorescently labeled antibodies.

  • Flow cytometry: Antibodies conjugated to fluorescent molecules specifically bind to cell surface markers or intracellular proteins, allowing for the analysis and sorting of cells based on antigen profiles.

Therapeutic applications: Fab

Fab fragments are used in therapeutic applications such as antibody-based therapies for neutralizing toxins, targeting specific antigens, or modulating immune responses. They are engineered for enhanced tissue penetration and reduced immunogenicity, making them suitable for conditions like autoimmune diseases, cancer, and infectious diseases.

Therapeutic applications: F(ab’)2

F(ab')2 fragments may potentially be employed in therapies targeting cell surface receptors, tumor antigens, or infectious agents, providing improved potency and specificity.

Therapeutic applications: Fc

Fc fragments play crucial roles in therapeutic applications involving antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and immune complex formation. They are utilized in antibody-based therapies for enhancing immune responses, eliminating infected or cancerous cells, or modulating inflammatory processes.

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