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Antibody Fragmentation

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.

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Overview of Antibody Production and Purification

Introduction

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.

Note:

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 structure and cleavage sites for fragmentation
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.

 

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Antibody Structure and
Classes of Immunoglobulins

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.

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Ready-made, fragment-specific secondary antibodies (2°Ab) are commercially available. Properties and uses of these antibodies are described in Tech Tip #59: Choosing a secondary antibody: A guide to fragment specificity

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.

Types of antibody fragments
 

F(ab')2 Fragments

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' Fragments

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 Fragments

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 Fragments

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" Fragments

"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 Fragments

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.
Antibody Fab preparation by papain digestion and fragmentation.

Papain:
• 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.

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Papain Agarose Resin

Fab Preparation Kits

Protein A Resins and Kits

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.

Antibody F(ab')2 preparation by pepsin digestion and fragmentation.
Antibody F(ab')2 preparation by pepsin digestion and fragmentation.

Pepsin:
• Acid-type protease
• MW 35,000
• Isoelectric point pI = 11
• pH optimum 1 (1 to 5)
• A280 at 1% = 14.7

F(ab')2 can be separated by mild reduction into two sulfhydryl-containing, univalent Fab' fragments. The advantage of Fab' fragments is that they can be conjugated to detectable labels directly through their sulfhydryl groups, ensuring that the active binding site remains unhindered and active. Use 2-Mercaptoethylamine•HCl (2-MEA) for mild reduction of F(ab')2 fragments. The free sulfhydryls of each Fab' can be targeted for conjugation, or they can be blocked with an alkylating reagent, such as N-Ethylmaleimide (NEM) to prevent re-formation of the F(ab')2.

Immobilized pepsin (Pepsin Agarose Resin) can be substituted for free pepsin in any application. Use of pepsin resin allows one to control digestion by quickly removing the enzyme from the sample to stop the reaction. This eliminates the need to develop an ion exchange method to separate the fragments from the protease. Also, immobilization increases the stability of the pepsin against heat denaturation and autolysis, resulting in longer maintenance of activity. Our Pierce F(ab')2 Preparation Kits have been optimized for human IgG digestions. The kits also can be used successfully for rabbit and mouse IgG digestions.

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2-Mercaptoethylamine

N-Ethylmaleimide

Pepsin Agarose Resin

F(ab')2 Preparation Kits

Protein A Resins and Kits

Ficin Digestion: Mouse IgG1 Fragments

Ficin is a thiol protease that can digest mouse monoclonal IgG1 into either F(ab')2 or Fab fragments, depending on the concentration of cysteine included. Ficin will generate F(ab')2 in the presence of 4mM cysteine. Fab fragments result with ficin in the presence of 25mM cysteine.

Mouse IgG1 Fab and F(ab')2 preparation by ficin digestion and fragmentation.
Mouse IgG1 Fab and F(ab')2 preparation by ficin digestion and fragmentation.

Ficin:
• Thiol-type protease
• MW 26,000
• Isoelectric point pI = 1.5
• pH optimum 6.5 (4 to 9.5)
• A280 at 1% = 21

Ficin cleavage produces F(ab')2 fragments of nearly identical size to those obtained from IgG by pepsin but with immunoreactivities and affinities comparable to those of intact IgG1 antibody (3). By increasing the concentration of cysteine activator, Fab antigen-binding fragments can be generated (4). The integrity of the resultant antigen-binding fragments is aided by the neutral pH conditions of the ficin digestion. Ficin is the preferred protease for production of fragments of murine monoclonal IgG1. Although F(ab')2 can be generated from mouse IgG1 using pre-activated papain (5), efficiency and reproducibility with papain are difficult to obtain (6).

Immobilized ficin (Ficin Agarose Resin) enables better control of the digestion reaction than free ficin, as it results in antibody fragments that are free of autodigestion products. In addition, use of ficin resin allows complete and rapid removal of ficin from the antibody fragment products. Our Pierce IgG1 Fab and F(ab')2 Preparation Kit was developed to allow gentle production and purification of either Fab or F(ab')2 fragments from intact murine IgG1 antibodies. The type of fragment produced is controlled by the specific concentration of cysteine activator used during the digestion.

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Ficin Agarose Resin

Mouse IgG1 Fab and F(ab')2 Preparation Kits

Protein A Resins and Kits

IgM Fragmentation

The large size of IgM creates difficulties in applications in which IgM is used for in vitro experiments. For example, intact IgM does not effectively penetrate tissues for immunohistochemical studies. Thus, it is necessary to produce smaller, active fragments for these types of applications. Also, because IgM molecules have difficulty permeating cell membranes, they are not ideal for use in vivo. Fragments are cleared more rapidly than intact IgM.

F(ab')2, Fab', Fab and Fv fragments can produced from IgM function in much the same way as these same types of fragments from IgG. However, compared to those in IgG, individual antigen-binding sites in IgM generally have lower binding affinities, which normally are compensated in the complete IgM by its pentameric form. Thus, because F(ab')2 fragments are divalent, and they may be a superior alternative to Fab fragments for antibodies with low affinity. F(ab')2 fragments have higher avidity than the Fab and Fab' fragments. F(ab')2 fragments can precipitate antigen. Fab and Fab' are univalent molecules that cannot precipitate antigen. Fab and Fab' fragments have a decreased binding strength, and normally stable antigen-antibody complexes may dissociate during washes in certain applications.

Methods of IgM antibody fragment preparation.
Methods of IgM antibody fragment preparation.

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Antibody Structure and
Classes of Immunoglobulins

Each species of IgM reacts differently to enzymatic cleavage and reduction. For example, mouse and human IgM differ structurally in the manner in which the monomers are linked to give the pentameric form, primarily as a result of differences in the location of disulfides between the monomers (7). Also, oligosaccharide components, which may hinder enzymatic cleavage, vary among species. Therefore, optimal digestion and reduction conditions for one species may not be effective for another.

Fragmentation of IgM by proteolyic enzymes proceeds differently from IgG fragmentation. These changes are related to differences in structure. The heavy (µ) chains are folded into multiple globular domains, and IgM has a carbohydrate-rich Cµ2 domain in place of the proline-rich hinge-region of IgG. Because of these features, papain produces heterogeneous fragments from IgM.

Pepsin (see discussion above) can be used to produce F(ab')2, Fab and Fv fragments from IgM. Many methods have been developed that use pepsin to produce different IgM fragments from different species (8).

Trypsin is a serine protease that reacts optimally at pH 8.0. In general, increasing the enzyme:substrate ratio and/or the temperature will increase the rate of digestion. Trypsin can generate F(ab')2, Fab, "IgG"-type and Fc5µ fragments from IgM. Fragmentation was studied using trypsin with and without urea pre-treatment (8). Urea alters the susceptibility of the domains to digestion and produces different fragments from those digested in aqueous buffer. Many other procedures have been developed to digest IgM using trypsin (9).

Controlled reduction can be performed using 2-Mercaptoethylamine•HCl (2-MEA) to obtain "IgG"-type and/or reduced IgG ("rIgG") varied proportions, depending upon reduction time and/or temperature (10). Partial reduction of mouse IgM also produces an inverted "IgG"-type fragment.

Trypsin:
• Serine-type protease
• MW 24,000
• Isoelectric point pI = 1.5
• pH optimum 8
• A280 at 1% = 14.3

 

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Trypsin Agarose Resin

Pepsin Agarose Resin

2-Mercaptoethylamine

References

  1. Coulter, A. and Harris, R. (1983). J. Immunol. Meth. 59, 199-203.
  2. Goding, J. (1983). Monoclonal Antibodies: Principles and Practice. Academic Press Inc., London, U.K.
  3. Mariani, M., et al. (1991). Mol. Immunol. 28, 69-77.
  4. Sykaluk, L. (1992). Pierce Chemical Company, unpublished results.
  5. Buguslawski, S.J., et al. (1989). J. Immunol. Meth. 120, 51-56.
  6. Milenic, D.E., et al. (1989). J. Immunol. Meth. 120, 71-83.
  7. Milstein, C.P., et al. (1975). Biochem. J. 151, 615-624.
  8. Beale, D. and Van Dort, T. (1982). Comp. Biochem. Physiol. 71B(3), 475-482.
  9. Plaut, A.G. and Tomasi, Jr., T.B. (1970). Proc. Natl. Acad. Sci. USA 65(2), 318-322.
  10. Bevan, M.J., et al. (1972). Progr. Biophys. Molec. Biol. 25, 131.
 
Written and/or reviewed by Douglas Hayworth, Ph.D.

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