Fragmentation of Immunoglobulin G

Sarah M. Andrew1, Julie A. Titus2

1 Lancaster University, Lancaster, United Kingdom, 2 National 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

  • 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
  • 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)
  • recipeDigestion 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)
  • recipeDigestion buffer (see recipe)
  • ≥1 mg/ml IgG (≥5 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)
  • recipeAcetate buffer, pH 4.0 and 4.5 (see recipe)
  • 0.1 mg/ml pepsin (Sigma) in recipepH 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
  • ≥1 mg/ml purified IgG (unit 2.7)
  • recipeAcetate buffer at appropriate pH (see protocol 3 and recipe)
  • 0.1 mg/ml pepsin in recipeacetate buffer at appropriate pH (see 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

  • 10 mg IgG (unit 2.7) in 2 to 5 ml PBS
  • recipeAcetate/EDTA buffer (see recipe)
  • 2 mg/ml papain in recipeacetate/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

  • 10 mg mouse monoclonal IgG 1 (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

Videos

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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