Fragmentation of Immunoglobulin G

Sarah M. Andrew1, Julie A. Titus2

1 Chester College, Chester, 2 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 16.4
DOI:  10.1002/0471143030.cb1604s17
Online Posting Date:  February, 2003
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Abstract

For some purposes, fragments of the IgG molecule are preferred. The Fc portion is useful for studies of biological effect–binding to the Fc receptor, mediating antibody‐dependent cellular cytotoxicity, and complement fixation. The bivalent F(ab')2 produced by digestion with pepsin and the monovalent Fab produced by digestion with papain are useful for studies based on the interaction between antibody binding site(s) with antigen. This unit also describes alternative methods for preparing F(ab')2 fragments.

     
 
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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 16.3) in PBS (unit 16.3)
  • Papain (2× crystallized suspension; Sigma)
  • Digestion buffer (see recipe)
  • 0.3 M iodoacetamide (prepare fresh from crystalline material; Sigma) in PBS
  • PBS (unit 16.3)
  • Additional reagents and equipment for nonreducing gel electrophoresis (unit 16.5) and dialysis of proteins ( appendix 3C)

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

  Materials
  • Papain (2× recrystallized suspension; Sigma)
  • Digestion buffer (see recipe)
  • ≥1 mg/ml IgG (≥5 mg total; unit 16.3) in PBS
  • Iodoacetamide crystals
  • PBS, pH 8.0 (unit 16.3)
  • 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 3C), column chromatography (unit 16.3 & appendix 3A), concentrating protein solutions ( appendix 3C), and nonreducing gel electrophoresis (unit 6.5)

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

  Materials
  • 3 mg/ml purified IgG (unit 16.3)
  • 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 (unit 16.3)
  • Additional reagents and equipment for protein dialysis ( appendix 3C) and nonreducing gel electrophoresis (unit 6.5)

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

  Materials
  • ≥1 mg/ml purified IgG (unit 16.3)
  • Acetate buffer at appropriate pH (see protocol 3 and recipe)
  • 0.1 mg/ml pepsin in acetate buffer at appropriate pH (see protocol 3)
  • 2 M Tris base
  • PBS, pH 8.0 (unit 16.3)
  • 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 3C), concentrating protein solutions ( appendix 3C), column chromatography (unit 16.3 & appendix 3A), and reducing and nonreducing gel electrophoresis (unit 6.5)

Alternate Protocol 1: Fragmentation of IgG Using Preactivated Papain

  • 10 mg IgG (unit 16.3) 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 3C), column chromatography (unit 16.3 & appendix 3A), and SDS‐PAGE (unit 6.1)

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

  • 10 mg mouse monoclonal IgG 1 (unit 16.3) 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 (unit 16.3)
  • Additional reagents and equipment for protein dialysis ( appendix 3C) and column chromatography (unit 16.3 & appendix 3A)
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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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