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Fragmentation of Immunoglobulin G

Sarah M. Andrew1,  Julie A. Titus2

1Lancaster University, Lancaster, United Kingdom, United Kingdom
2National Cancer Institute, Bethesda, Maryland


Publication Name: 
Current Protocols in Immunology
Unit Number: 
Unit 2.8
DOI: 
10.1002/0471142735.im0208s21
Online Posting Date: 
May, 2001
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Abstract

This unit describes procedures for fragmentation of IgG to the monovalent Fab fragment using papain digestion, and to the bivalent F(ab¢)2 fragment by pepsin digestion. Alternative methods of fragmentation to F(ab¢)2 include use of papain that is preactivated with cysteine and use of the enzyme ficin. These alternate methods are particularly useful for mouse IgG1 antibodies.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Pilot Fragmentation of IgG to Fab Using Papain
  • Basic Protocol 2: Large-Scale Fragmentation of IgG to Fab Using Papain
  • Basic Protocol 3: Pilot Fragmentation of IgG to F(ab¢)2 Using Pepsin
  • Basic Protocol 4: Large-Scale Fragmentation of IgG to F(ab¢)2 Using Pepsin
  • Alternate Protocol 1: Fragmentation of IgG Using Preactivated Papain
  • Alternate Protocol 2: Fragmentation of Mouse IgG1 to F(ab¢)2 Using Ficin
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
     
 
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Materials

Basic Protocol 1: Pilot Fragmentation of IgG to Fab Using Papain

 Materials
  • 2 mg/ml purified IgG (unit 2.7) in PBS (appendix 2A)
  • Papain (2× crystallized suspension; Sigma)
  • Digestion buffer (see recipe)
  • 0.3 M iodoacetamide (prepare fresh from crystalline material; Sigma) in PBS
  • PBS (appendix 2A)
  • Additional reagents and equipment for SDS-PAGE (unit 8.4) and dialysis of proteins (appendix 3H)

Basic Protocol 2: Large-Scale Fragmentation of IgG to Fab Using Papain

 Materials
  • Papain (2× recrystallized suspension; Sigma)
  • Digestion buffer (see recipe)
  • ³1 mg/ml IgG (³5 mg total; unit 2.7) in PBS
  • Iodoacetamide crystals
  • PBS, pH 8.0 (appendix 2A)
  • Protein A–Sepharose CL-4B
  • Sephacryl S-200 Superfine (Pharmacia Biotech)
  • 5 × 100–mm column (Bio-Rad)
  • 26 × 900–mm column (Pharmacia Biotech)
  • Additional reagents and equipment for protein dialysis (appendix 3H), column chromatography (unit 2.7 & appendix 3I), concentrating protein solutions (appendix 3H), and SDS-PAGE (unit 8.4)

Basic Protocol 3: Pilot Fragmentation of IgG to F(ab¢)2 Using Pepsin

 Materials
  • 3 mg/ml purified IgG (unit 2.7)
  • Acetate buffer, pH 4.0 and 4.5 (see recipe)
  • 0.1 mg/ml pepsin (Sigma) in pH 4.0 and pH 4.5 acetate buffers
  • 2 M Tris base
  • PBS (appendix 2A)
  • Additional reagents and equipment for protein dialysis (appendix 3H) and SDS-PAGE (unit 8.4)

Basic Protocol 4: Large-Scale Fragmentation of IgG to F(ab¢)2 Using Pepsin

 Materials
  • ³1 mg/ml purified IgG (unit 2.7)
  • Acetate buffer at appropriate pH (see Basic Protocol 3 and recipe)
  • 0.1 mg/ml pepsin in acetate buffer at appropriate pH (see Basic Protocol 3)
  • 2 M Tris base
  • PBS, pH 8.0 (appendix 2A)
  • Protein A–Sepharose CL-4B
  • Sephacryl S-200 Superfine (Pharmacia Biotech)
  • 5 × 100–mm column (Bio-Rad)
  • 26 × 900–mm column (Pharmacia Biotech)
  • Additional reagents and equipment for protein dialysis (appendix 3H), concentrating protein solutions (appendix 3H), column chromatography (unit 2.7 & appendix 3I), and SDS-PAGE (unit 8.4)

Alternate Protocol 1: Fragmentation of IgG Using Preactivated Papain

 Additional Materials (also see Basic Protocol 4)
  • 10 mg IgG (unit 2.7) in 2 to 5 ml PBS
  • Acetate/EDTA buffer (see recipe)
  • 2 mg/ml papain in acetate/EDTA buffer
  • 0.05 M cysteine (free base, crystalline; Sigma)
  • Iodoacetamide crystals
  • PD-10 column (Pharmacia Biotech)
  • 26 × 900–mm column
  • Additional reagents and equipment for protein dialysis and concentration (appendix 3H), column chromatography (unit 2.7 & appendix 3I), and SDS-PAGE (unit 8.4)

Alternate Protocol 2: Fragmentation of Mouse IgG1 to F(ab¢)2 Using Ficin

 Additional Materials (also see Basic Protocol 4)
  • 10 mg mouse monoclonal IgG1 (unit 2.7) in 2 to 5 ml PBS
  • 50 mM Tris/2 mM EDTA, pH 7
  • Ficin solution: 1 mg/ml ficin (Sigma) in 50 mM Tris/2 mM EDTA, pH 7
  • Cysteine
  • 100 mM N-ethylmaleimide (Sigma)
  • PBS (appendix 2A)
  • Additional reagents and equipment for protein dialysis (appendix 3H) and column chromatography (unit 2.7 & appendix 3I)
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Figures

  • Figure 2.8.1
    Proteolytic digestion fragments of immunoglobulins. Structural domains of the variable and constant regions of the light chain (VL, CL) and heavy chain (VH, CH1, CH2, CH3), as well as interchain disulfide bonds (S-S), are indicated. Monovalent fragments of IgG produced by papain digestion are known as Fab, while bivalent papain fragments are F(ab)2. F(ab¢)2 refers to the bivalent fragments of IgG produced by pepsin digestion; those derived from IgM are called F(ab¢)2µ. Bivalent subunits of IgM with both Fab and Fc are known as IgMs (subunit), and fragments of IgG containing a single antibody-binding site and an intact Fc portion are Fab/c.

    View Image

Literature Cited

 Literature Cited
    Hellstrom, K.E. and Hellstrom, I. 1985. Monoclonal anti-melanoma antibodies and their possible use. In Monoclonal Antibodies for Cancer Detection and Therapy (R.W. Baldwin and V.S. Byers, eds.) pp.17-51. Academic Press, London.
    Mariani, M., Cauragra, M., Tarditi, L., and Seccariani, E. 1991. A new enzymatic method to obtain high-yield F(ab¢)2 suitable for clinical use from mouse IgG1. Mol. Immunol. 28:69-77.
    Overall, C.M. 1987. A microtechnique for dialysis of small volume solutions with quantitative recoveries. Anal. Biochem. 165:208.
    Parham, P. 1983. On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice. J. Immunol. 131:2895-2902.
    Parham, P. 1986. Preparation and purification of active fragments from mouse monoclonal antibodies. In Handbook of Experimental Immunology, Vol.1: Immunochemistry ( D.M. Wier, ed.) pp.14.1-14.23. Blackwell Scientific, Oxford.
    Parham, P., Androlewicz, M.J., Brodsky, F.M., Holmes, N.J., and Ways, J.P. 1982. Monoclonal antibodies: Purification, fragmentation, and application to structural and functional studies of class I MHC antigens. J. Immunol. Methods 53:133-147.
 Key Reference
    Parham, 1983. See above.

A lucid and comprehensive guide to the production of fragments from mouse monoclonal antibodies.

     
 
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