Antibody Fragments
SmartEnzymes for Antibody Fragment Generation
Antibody fragments are important tools in many scientific areas utilizing the unique antigen binding properties of the antibody, but in a smaller format. The generation of fragments can be obtained using specific proteolytic digestion and affinity purification. Many of the Genovis SmartEnzymes can be used for both small scale and preparative generation of fragments used in a range of applications discussed here.
Antibody Fragments Explained
The IgG antibody isotype is responsible for the majority of antibody-based immunity against pathogens and therefore highly favored for the development of biopharmaceuticals. The intact IgG molecule is a large plasma protein (150 kDa) consisting of two heavy chains (50 kDa) and two light chains (25 kDa). The heavy and light chains are in turn held together by a combination of non-covalent interactions and covalent interchain disulfide bonds to form a globular protein structure. For human IgG, the number of inter-chain disulfide bridges range from two in IgG1 and IgG4, four in IgG2 and eleven in IgG3. The molecular complexity of the intact antibody can be reduced by proteolytic digestion to generate smaller fragments that are advantageous in many experimental and analytical workflows. The nomenclature for antibody fragments and the discrete characteristics of each fragments is summarized in the schematic illustration below (Fig. 1).
Figure 1. Schematic illustration of a general IgG molecule and corresponding fragments.
F(ab’)2 – A 110 kDa antibody fragment with two antigen binding Fab domains with intact hinge region held together by the disulphide bridges
Fab’ – The antigen binding domain with reduced disulphide hinge region, mass around 55 kDa
Fab – An antigen binding domain without the hinge region, mass 50 kDa
Fc – A 50 kDa fragment of the antibody involved in interactions with Fc receptors and thus effector functions on immune cells. Contains a conserved glycosylation site at Asn297.
Fc/2 – A single heavy chain of the Fc.
Enzymes to Generate Antibody Fragments
The IgG hinge region is readily accessible to proteolytic attack by enzymes. The traditional enzymes used to generate fragments at a preparative scale include papain, ficin and pepsin. Although standardized, these enzymes are often associated with fragment heterogeneity, low yields and loss of immunoreactivity due to digestion at a low pH. These aspects result in time-consuming reaction condition optimizations and complex workflows. The SmartEnzymes IgG proteases can overcome such limitations and allow the generation of homogenous antibody fragments from several different species and subclasses of IgG, shown in the table below.
Applications of Antibody Fragments
Once digested, the antibody can be analyzed at subunit level in middle-level LC-MS workflows for antibody characterization. The antibody fragments can also be used for their specificity as valuable tools in applications such as protein purification, binding studies and as analytical reagents in imaging or bioassays. In certain applications, it is also preferred to isolate the different fragments to avoid disturbance from other parts of the antibody molecule. For instance, the antigen-binding F(ab’)2 or Fab fragments may be isolated to avoid unwanted interactions between the Fc fragment and Fc receptors (FcRs) in cell and tissue-based applications, both in vivo and in vitro. Further applications for isolated F(ab’)2 and Fc fragments include:
- To eliminate effector functions associated with Fc
- Reduction of non-specific binding from Fc
- Increased sensitivity in antigen detection by reduced steric hindrance from large epitopes
- Improved penetration into tissues for improved staining in immunohistochemistry (IHC) and imaging
- Lower immunogenicity than intact antibody for in vivo experiments
- Less complex system for structural analysis by using crystallography and NMR
- Study monovalent binding of Fab fragments to the antigen