Antibodies are powerful tools for protein and molecular detection and purification. Although whole antibodies (usually IgG or IgM) are ideal for most immunoassay applications, the performances of certain procedures are enhanced by using antibody fragments, such as Fab and F(ab')2. This article reviews the benefits, types and methods for preparing antibody fragments.
Sometimes it is useful to study or make use of the activity of one portion of an immunoglobulin without interference from other portions of the molecule. It is possible to selectively cleave the immunoglobulin molecule into fragments that have discrete characteristics. Antibody fragmentation is accomplished using reducing agents and proteases that digest or cleave certain portions of the immunoglobulin protein structure. Although fragmentation of all immunoglobulin classes is possible, only procedures for fragmentation of mouse, rabbit, and human IgG and IgM have been well characterized.
The two groups of antibody fragments of primary interest are (a) antigen-binding fragments such as Fab and (b) class-defining fragments such as Fc that do not bind antigen. Several types of antigen-binding fragments are possible, but each contains at least the variable regions of both heavy and light immunoglobulin chains (VH and VL, respectively) held together (usually by disulfide bonds) so as to preserve the antibody-binding site. Fc fragments consist of the heavy chain constant region (Fc region) of an immunoglobulin and mediate cellular effector functions.
Antibody fragmentation is somewhat laborious, requires optimization of enzyme-mediated digestion of the protein and necessitates an ample supply of antibody (e.g., 10mg) to make it reasonably efficient. For these reasons, fragmentation is usually performed only when the antibody of interest is available in large quantity and the particular application demands it.
- Primary antibodies (1°Ab) are seldom offered commercially as ready-made fragments because there is limited demand for any given item. For this reason, except with custom antibody production, fragmentation is an activity for each individual laboratory to perform for its specific needs.
Antibody IgG structure and cleavage sites for fragmentation. Useful antibody fragments, including half-IgG, Fab, F(ab')2 and Fc, can be produced by reduction of hinge-region disulfides or digestion with papain, pepsin or ficin proteolytic enzymes.
Advantages of antibody fragments
Because of their smaller size as functional components of the whole molecule, antibody fragments offer several advantages over intact antibodies for use in certain immunochemical techniques and experimental applications:
- Reduced nonspecific binding from Fc interactions (many cells have receptors that bind the Fc region)
- Ability to control Fc-binding to Protein A or Protein G in experiments involving immunoprecipitation and Western blotting
- More efficient penetration of tissue sections, resulting in improved staining in immunohistochemistry (IHC)
- Potentially higher sensitivity in antigen detection in solid phase applications as a result of reduced steric hindrance from large protein epitopes
- Elimination of Fc-associated effector functions (e.g,. complement fixation) in antigen-antibody binding studies
- Simpler system for studying the structural basis for immune recognition using X-ray crystallography or NMR
- Lower immunogenicity than intact antibody for experiments in vivo
Types of antibody fragments
F(ab')2, Fab, Fab' and Fv are antigen-binding fragments that can be generated from the variable region of IgG and IgM. These antigen-binding fragments vary in size (MW), valency and Fc content. Fc fragments are generated entirely from the heavy chain constant region of an immunoglobulin. These and several additional unique fragment structures can be generated from pentameric IgM, including an "IgG"-type fragment, an inverted "IgG"-type fragment, and a pentameric Fc fragment.
The names (nomenclature) and structures of typical IgG fragments are illustrated in the following diagram and summarized below.
F(ab')2 (110,000 daltons) fragments contain two antigen-binding regions joined at the hinge through disulfides. This fragment is void of most, but not all, of the Fc region.
Fab' (55,000 daltons) fragments can be formed by the reduction of F(ab')2 fragments. The Fab' fragment contains a free sulfhydryl group that may be alkylated or utilized in conjugation with an enzyme, toxin or other protein of interest. Fab' is derived from F(ab')2; therefore, it may contain a small portion of Fc.
Fab (50,000 daltons) is a monovalent fragment that is produced from IgG and IgM, consisting of the VH, CH1 and VL, CL regions, linked by an intramolecular disulfide bond.
Fv (25,000 daltons) is the smallest fragment produced from IgG and IgM that contains a complete antigen-binding site. Fv fragments have the same binding properties and similar three-dimensional binding characteristics as Fab. The VH and VL chains of the Fv fragments are held together by non-covalent interactions. These chains tend to dissociate upon dilution, so methods have been developed to cross-link the chains through glutaraldehyde, intermolecular disulfides or a peptide linker.
"rIgG" refers to reduced IgG (75,000 daltons) or half-IgG. It is the product of selectively reducing just the hinge-region disulfide bonds. Although several disulfide bonds occur in IgG, those in the hinge-region are most accessible and easiest to reduce, especially with mild reducing agents like 2-mercaptoethylamine (2-MEA). Half-IgG are frequently prepared for the purpose of targeting the exposing hinge-region sulfhydryl groups that can be targeted for conjugation, either antibody immobilization or enzyme labeling.
Fc (50,000 daltons) fragments contain the CH2 and CH3 region and part of the hinge region held together by one or more disulfides and noncovalent interactions. Fc and Fc5µ fragments are produced from fragmentation of IgG and IgM, respectively. The term Fc is derived from the ability of these antibody fragments to crystallize. Fc fragments are generated entirely from the heavy chain constant region of an immunoglobulin. The Fc fragment cannot bind antigen, but it is responsible for the effector functions of antibodies, such as complement fixation.
IgG—Preparing Fab, F(ab')2 and Fc fragments
The hinge region of an immunoglobulin monomer (IgG) is readily accessible to proteolytic attack by enzymes. Cleavage at this point produces F(ab')2 or Fab fragments and the Fc fragment. The Fc fragment may remain intact or become further degraded, depending upon the enzyme and conditions used. Proteolytic IgG fragmentation using three different enzymes is discussed below. Traditionally, proteolysis was accomplished in solution using free enzyme. We have developed immobilized enzyme products that enable better control of digestion and efficient separation of reaction-products from the protease. Thus, the diagrams featured below refer to enzyme "resins." Most procedures also include Protein A resin antibody purification steps to separate Fab and Fc fragments.
Papain digestion: Fab from IgG
Papain is a nonspecific, thiol-endopeptidase that has a sulfhydryl group in the active site, which must be in the reduced form for activity. When IgG molecules are incubated with papain in the presence of a reducing agent, one or more peptide bonds in the hinge region are split, producing three fragments of similar size: two Fab fragment and one Fc fragment (1). When Fc fragments are of interest, papain is the enzyme of choice because it yields an intact 50,000-dalton Fc fragment.
Antibody Fab preparation by papain digestion and fragmentation.
- Thiol-type protease
- MW 23,000
- Isoelectric point pI = 1.5
- pH optimum 6.5 (4 to 9.5)
- A280 at 1% = 25
Papain is primarily used to generate Fab fragments, but it also can be used to generate F(ab')2 fragments (2). To prepare F(ab')2 fragments, the papain is first activated with 10mM cysteine. The excess cysteine is then removed by gel filtration. If no cysteine is present during papain digestion, F(ab')2 fragments can be generated. These fragments are often inconsistent, and reproducibility can be a problem. If the cysteine is not completely removed, overdigestion can be a problem (2).
Crystalline papain is often used for the digestion of IgG; however, it is prone to autodigestion. Mercuripapain, which is less prone to autodigestion than crystalline papain, can be used; however, both of these non-immobilized enzymes require an oxidant to terminate digestion. Immobilized papain (i.e., Papain Agarose Resin) is the preferred reagent because it allows for easy control of the digestion reaction and quick removal of enzyme from the digestion products following incubation. Thus, there is no need to develop an ion exchange method for separating the fragments from the enzyme. The use of immobilized papain alsos prevent formation of antibody-enzyme adducts, which can occur when using the soluble form of sulfhydryl proteases (such as papain). These adducts can be detrimental to fragments in the presence of reductants.
Immobilization also increases stability of the enzyme against heat denaturation and autolysis and results in longer maintenance of activity. Regeneration and reuse of papain resin, which decreases costs. Cleavage can be regulated by digestion time or flow rate through a column, yielding reproducible digests. Our Pierce Fab Preparation Kits are been optimized for human IgG digestions. The kits also can be used successfully for mouse and rabbit IgG digestions, and suggestions on how to vary the protocols for other species of IgG are provided with the kit. The procedures requires that the IgG is able to be bound by Protein A, as it is used to separate Fc from Fab fragments.
Pepsin digestion: F(ab')2 from IgG
Pepsin is a nonspecific endopeptidase that is active only at acid pH. It is irreversibly denatured at neutral or alkaline pH. Digestion by the enzyme pepsin normally produces one F(ab')2 fragment and numerous small peptides of the Fc portion. The resulting F(ab')2 fragment is composed of two disulfide-connected Fab units. The Fc fragment is extensively degraded, and its small fragments can be separated from F(ab')2 by dialysis, gel filtration or ion exchange chromatography.