Prepare and purify Fab or F(ab')2 fragments from mouse IgG1 antibodies.

The Mouse IgG1 Fab and F(ab')2 Preparation Kits use immobilized ficin protease to generate Fab or F(ab')2 fragments from mouse IgG1. Both regular and micro-scale kits provide include all the reagents and accessories necessary to produce either Fab or F(ab')2 fragments depending on the concentration of cysteine that is used in the digestion reaction. When incubated with whole IgG1 and 1-4 mM cysteine, ficin produces F(ab')2 fragments. When 25 mM cysteine is used, ficin digestion produces Fab fragments.

The antibody digestion reaction is performed in convenient disposable spin columns that allow efficient removal of the immobilized protease and maximum recovery of the IgG fragments. Also included in the kits are Thermo Scientific NAb Protein A Spin columns and buffers to efficiently purify the resulting fragments. Protein A binds the Fc fragments and undigested IgG, allowing the pure Fab fragments to be recovered in the flow-through fraction. The kits also include Thermo Scientific Zeba Desalting Spin Columns for preparing the IgG sample quickly without dilution instead of utilizing time-consuming dialysis steps.

Figure 1. Digestion scheme for preparing F(ab')2 fragments from mouse IgG1 antibodies. Depending on the concentration of cysteine used in the digestion buffer, primarily F(ab')2 or Fab can be made.

Why make Antibody Fragments?

Because of their smaller size as functional components of the whole molecule, Fab and F(ab')2 antibody fragments offer several advantages over intact antibodies for use in certain immunochemical techniques and experimental applications. Fab fragments have the advantage of being the smallest antigen-binding fragments, but they may have reduced effectiveness in certain applications because of their monovalent structure. F(ab')2 fragments derive particular utility from having no Fc domain and somewhat smaller size than whole IgG while also maintaining avidity and other properties of divalent structure. Some general advantages of Fab and F(ab')2 fragments include...

• Reduced nonspecific binding that results from Fc interactions (many cells have receptors that bind the Fc region)
• Ability to control binding to Protein A in experiments involving immunoprecipitation and Western blotting
• More efficient penetration of tissue sections, resulting in improved staining for immunohistochemical applications
• 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 study the structural basis for immune recognition using X-ray cyrstallography or NMR
• Lower immunogenicity that intact antibody, an advantage for experiments in vivo

Highlights

• Enzyme-free digestion products – Immobilized Ficin (beaded agarose resin) provides for control of the digestion reaction and complete removal of resulting antibody fragments from the proteolytic enzyme
• Optimized for mouse IgG1 – the kit procedure is optimized for mouse IgG1; the only effective method for this IgG species and subclass (see Related Products for Fab and F(ab')2 preparation kits that are optimized for other species and subclasses of IgG)
• Provides ready-to-use antibody fragments – digestion and final recovery of purified Fab or F(ab')2 fragments occurs in neutral pH sodium phosphate buffer, suitable for storage or immediate use in typical applications
• High capacity – kits provide for digestion and purification from as much as 4 mg of IgG at a time. Use the standard size kit for fragmenting 0.25 to 4 mg IgG, and the micro kit for 25 to 250 µg IgG.
• Complete – kits include all reagents needed to prepare and purify antibody fragments
• Fast – spin format greatly reduces sample processing time

Figure 2. Analysis of various experimental fractions by non-reducing SDS-PAGE (8 - 16%). Each well was loaded with 2 µl of protein.

Related Products
Fab Preparation Kit – for other species and subclasses besides mouse IgG1
F(ab')2 Preparation Kit – for other species and subclasses besides mouse IgG1
IgM Fragmentation Kit

References

1. Beale, D. (1987). Molecular fragmentations. Some applications in immunology. Dev Comp Immunol 11:287-96.
2. Boguslawski, S.J., et al. (1989). Improved procedure for preparation of F(ab')2 fragments of mouse IgGs by papain digestion. J. Immunol. Meth. 120:51-56.
3. Kerbel, R.S. and Elliot, B.E. (1983). Detection of Fc receptors. Method. Enzymol. 93:113-147.
4. Kulkami, P.N., et al. (1985). Conjugation of methotrexate to IgG antibodies and their F(ab')2 fragments and the effect of conjugated methotrexate on tumor growth in vivo. Cancer Immunol. Immunother. 19:211-14.
5. Mariani, M. et al. (1991). A new enzymatic method to obtain high-yield F(ab')2 suitable for clinical use from mouse IgG1. Mol. Immunol. 28:69-77.
6. Milenic, D.E., et al. (1989). Comparison of methods for the generation of immunoreactive fragments of a monoclonal antibody (B72.3) reactive with human carcinomas. J. Immunol. Meth. 120:71-83.
7. Nisonoff, A., et al. (1960). Properties of the major component of a peptic digest of rabbit antibody. Science 132:1770.
8. Parham, P.L., et al. (1982). Monoclonal antibodies: Purification, fragmentation and application to structural and functional studies of class I MHC antigens. J. Immunol. Meth. 53:133-73.
9. Parham, P. L. (1983). On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice. J. Immunol. 131:2895-2902.
10. Raychaudhuri, G., et al. (1985). Human IgG1 and its Fc fragment bind with different affinities to the Fc receptors on the human U937, HL-60, and ML-1 cell lines. Mol. Immunol. 22(9):1009-19.
11. Wilson, K.M., et al. (1991). A new enzymatic method to obtain high-yield F(ab')2 suitable for clinical use from mouse IgG1. Mol. Immunol. 28:69-77.