Purification of Monoclonal Antibodies

Steven A. Fuller1, Miyoko Takahashi1, John G.R. Hurrell1

1 Allelix Inc., Mississauga, Ontario
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 11.11
DOI:  10.1002/0471142727.mb1111s37
Online Posting Date:  May, 2001
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Abstract

The uses of monoclonal antibodies as enzyme conjugates and as immunoaffinity reagents require their purification from the crude ascites fluid. This unit provides protocols for purification using protein A‐Sepharose chromatography and affinity chromatography, which are superior to ammonium sulfate precipitation, gel filtration, or ion‐exchange chromatography for the preparation of contaminant‐free antibody.

     
 
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Table of Contents

  • Basic Protocol 1: Purification Using Protein A–Sepharose
  • Alternate Protocol 1: Alternative Buffer System for Protein A–Sepharose
  • Alternate Protocol 2: Purification by Antigen‐Sepharose and Anti‐Mouse Immunoglobulin‐Sepharose
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Purification Using Protein A–Sepharose

  Materials
  • Protein A–Sepharose CL‐4B ( Pharmacia)
  • recipeTris buffer, pH 8.6
  • Ascites fluid (unit 11.10)
  • recipeCitrate buffer, pH 5.5
  • recipeAcetate buffer, pH 4.3
  • recipeGlycine⋅Cl buffer, pH 2.3
  • recipeNeutralizing buffer, pH 7.7
  • 2.5‐cm (inner diameter) glass chromatography column
  • UV flowthrough detector or UV spectrophotometer and cuvette
  • Ultrafiltration cells and XM50 membranes (Amicon)
  • Additional reagents and equipment for ELISA screening (unit 11.4)

Alternate Protocol 1: Alternative Buffer System for Protein A–Sepharose

  Additional Materials
  • CNBr‐activated Sepharose 4B (Pharmacia)
  • 1 mM HCl
  • recipeCoupling buffer
  • Protein antigen (previously purified; see Chapter 10) or anti‐mouse immunoglobulin antibody (commercially available)
  • recipe1 M ethanolamine, pH 8.0
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Washing buffer
  • 60‐ or 150‐ml sintered glass funnel, medium porosity
  • 50‐ml conical plastic centrifuge tube
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Figures

Videos

Literature Cited

Literature Cited
   Ey, P.L., Prowse, S.J., and Jenkin, C.R. 1978. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A–Sepharose. Immunochemistry 15:429‐436.
   Oi, V.T. and Herzenberg, L.A. 1980. Immunoglobulin‐producing hybrid cell lines. In Selected Methods in Cellular Immunology (B.B. Mishell and S.M. Shiigi, eds.) pp. 351‐372. W.H. Freeman, San Francisco.
Key References
   Hurrell, J.G.R. ed. 1982. Monoclonal Hybridoma Antibodies: Techniques and Applications. CRC Press, Boca Raton, FL.
  These volumes present a large body of material on basic hybridoma methodology and detail the use of monoclonal antibodies in the study of hormones, structural proteins, viruses, parasites, and mammalian cell types.
   Langone, J.J. and Van Vunakis, H. eds., 1986. Immunological techniques, Part I: Hybridoma technology and monoclonal antibodies. Meth. Enzymol. 121:1‐947.
  Details the strategies and procedures for the preparation of monoclonal antibodies.
   Galfre, G. and Milstein, C. 1981. Preparation of monoclonal antibodies: Strategies and procedures. Meth. Enzymol. 73 (B):3‐46.
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