Purification 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 2.7
DOI:  10.1002/0471142735.im0207s21
Online Posting Date:  May, 2001
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Abstract

IgG can be purified, as described here, by ammonium sulfate precipitation followed by size‐exclusion (SE) chromatography. This is the least expensive option available for purification of antibodies. Protein A‐ and protein G‐affinity chromatography are the fastest methods for purifying antibodies, but they are not effective for all subclasses of rat antibody. A protocol for affinity chromatography using anti‐rat antibody is provided for purification of rat antibodies. Ion‐exchange (IEX) chromatography is described for purifying intact monoclonal and polyclonal antibodies and antibody fragments.

     
 
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Table of Contents

  • Purification and Fragmentation of Antibodies
  • Basic Protocol 1: Ammonium Sulfate Precipitation and Size‐Exclusion Chromatography
  • Basic Protocol 2: Affinity Chromatography Using Protein A–Sepharose
  • Alternate Protocol 1: Affinity Chromatography Using Protein G–sepharose
  • Alternate Protocol 2: Affinity Chromatography Using Anti–Rat κ Chain Monoclonal Antibody Coupled to Sepharose
  • Basic Protocol 3: DE52 Ion‐Exchange Chromatography with Tris⋅Cl
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Ammonium Sulfate Precipitation and Size‐Exclusion Chromatography

  Materials
  • Ascites fluid or MAb supernatant (unit 2.6)
  • PBS ( appendix 2A)
  • recipeSaturated ammonium sulfate (SAS; see recipe)
  • recipeBorate‐buffered saline (optional; see recipe)
  • Sephacryl S‐200 Superfine (Pharmacia Biotech)
  • PBS containing 0.02% sodium azide (optional)
  • Glass wool (Polysciences)
  • Sorvall centrifuge and SS‐34 rotor (or equivalent)
  • 26 × 900–mm column (Pharmacia Biotech)
  • Additional reagents and equipment for protein dialysis ( appendix 3H), column chromatography ( appendix 3I), concentrating proteins ( appendix 3H), and reducing and nonreducing PAGE (unit 8.4)

Basic Protocol 2: Affinity Chromatography Using Protein A–Sepharose

  Materials
  • Ascites fluid or MAb supernatant (unit 2.6)
  • PBS, pH 8.0 and pH 7.3 ( appendix 2A)
  • 1 M NaOH
  • Protein A–Sepharose CL‐4B, hydrated (Pharmacia Biotech or Sigma)
  • 0.1 M citric acid at pH appropriate for subclass of antibody (see step )
  • recipeBorate‐buffered saline(optional; see recipe)
  • 3 M potassium thiocyanate, filtered
  • Sorvall centrifuge and SS‐34 rotor (or equivalent)
  • 0.45‐µm filter
  • 1.5 × 10–cm column
  • HiTrap protein A column (Pharmacia Biotech or Sigma; optional)
  • Additional reagents and equipment for dialysis ( appendix 3H) and columnchromatography ( appendix 3A)

Alternate Protocol 1: Affinity Chromatography Using Protein G–sepharose

  • 0.1 M sodium acetate, pH 5.0
  • 0.1 M glycine⋅HCl, pH 2.8
  • HiTrap protein G column (Pharmacia Biotech or Sigma)

Alternate Protocol 2: Affinity Chromatography Using Anti–Rat κ Chain Monoclonal Antibody Coupled to Sepharose

  • Mouse anti‐rat κ MAb: MAR 18.5 (ATCC TIB 216) purified using protein A–Sepharose (see protocol 2)
  • CNBr–Sepharose CL‐4B (unit 8.3; Pharmacia Biotech)
  • Binding buffer: 0.05 M Tris⋅Cl/0.15 M NaCl/0.02% (w/v) NaN 3, pH 8.6
  • Crude rat antibody solution to be purified (MAb supernatant or ascites fluid; unit 2.6)
  • pH 7.0 elution buffer: 0.05 M sodium phosphate/0.15 M NaCl/0.02% (w/v)NaN 3, pH 7.0
  • pH 5.5 elution buffer: 0.05 M sodium citrate/0.15 M NaCl/0.02% (w/v)NaN 3, pH 5.5
  • pH 4.3 elution buffer: 0.5 M sodium acetate/0.15 M NaCl/0.02% (w/v) NaN 3, pH 4.3
  • pH 2.3 elution buffer: 0.5 M glycine/0.15 M NaCl/0.02% (w/v)NaN 3, pH 2.3
  • Additional reagents and equipment for preparation of antibody‐Sepharose (unit 8.3)

Basic Protocol 3: DE52 Ion‐Exchange Chromatography with Tris⋅Cl

  Materials
  • DE52 powder (Whatman)
  • recipe0.01 M Tris⋅Cl, pH 8.6 ( appendix 2A)
  • recipe0.5 M NaCl/0.01 M Tris⋅Cl, pH 8.6 (see recipe)
  • Antibody sample (ascites fluid, tissue culture supernatant, immune serum, or ammonium sulfate precipitate)
  • 1.5 × 50–cm column
  • Additional reagents and equipment for column chromatography ( appendix 3I), dialysis ( appendix 3H), and ELISA (unit 2.1) or SDS‐PAGE (unit 8.4)
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Figures

  •   FigureFigure 2.7.1 IEX chromatography elution profile from a DE52 column (1.5 × 50 cm). The IgG fractions from an ACA‐34 column were dialyzed against 0.01 M Tris⋅Cl, pH 8.6, and applied to the column. Ten fractions (12 ml) were collected using the starting buffer, then a linear gradient of 0 to 0.5 M NaCl in Tris⋅Cl, pH 8.6, was started. Fractions 19 to 22 contained pure IgG as determined by SDS‐PAGE ( UNIT).
  •   FigureFigure 2.7.2 IEX chromatography elution profile of Fab from two different antibodies using a DE52 column (1.5 × 50 cm). Elution conditions and gradient were the same as for the example in Figure . For MAb 315F6 (open circles), the Fab fragment eluted in the starting buffer and the Fc portion much later in the gradient. Fab fragments from MAb B72.3 (closed circles) eluted at the beginning of the gradient.

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Literature Cited

Literature Cited
   Akerstrom, B. and Bjorck, L. 1986. A physiochemical study of protein G molecule with unique immunoglobulin G–binding properties. J. Biol. Chem. 261:10240‐10247.
   Cooper, H.M. and Paterson, Y. 1989. Determination of the specific antibody titer. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 11.16.1‐11.16.18. John Wiley & Sons, New York.
Key References
   Hardy, R.R. 1986. Purification and characterization of monoclonal antibodies. In Handbook of Experimental Immunology, Vol. 1: Immunochemistry (D.M. Weir, ed.) pp. 13.1‐13.13. Blackwell Scientific, Oxford.
  An excellent and detailed review of methods for purifying antibodies that includes an extensive list of current literature.
   Lane, D. (ed.) 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
  The complete text on antibodies; everything you want to know and more.
   A Technical Guide to Antibody/Protein Purification. 1995. Pierce, Rockford, Ill.
  A highly informative supplement to the Pierce antibody‐purification kits.
  Monoclonal Antibody Purification Handbook. 1994. Pharmacia Biotech, Piscataway, N.J.
  This handbook contains the computer program MAb Assistant.
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