Fragmentation of Immunoglobulin M

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 2.10A
DOI:  10.1002/0471142735.im0210as38
Online Posting Date:  May, 2001
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Abstract

Fragmentation of IgM antibodies may be necessary because of the large molecular weight of the native molecule (900 kDa). IgMs fragments resemble IgG in size and structure, but they may have a decreased binding affinity. The Fc portion of IgM can have powerful biological effector functions such as complement activation. Because some T cells have receptors for IgM, it may be desirable to produce fragments of IgM for both cytotoxicity studies and for in vivo use. A protocol is presented for digestion of IgM with pepsin to produce F(ab′)2µ. The fragment can be reduced to produce the monovalent F(ab')u, if desired. IgM can also be reduced and alkylated in a single step, as described, using cysteine to produce IgMs, the bivalent monomer or subunit of IgM.

     
 
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Table of Contents

  • Basic Protocol 1: Fragmentation of IgM to F(ab′)2µ and F(ab′)µ
  • Basic Protocol 2: Fragmentation of IgM to IgMs Subunit
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Fragmentation of IgM to F(ab′)2µ and F(ab′)µ

  Materials
  • 3 to 5 mg/ml purified IgM antibody in PBS (unit 2.9)
  • Acetate buffer, pH 4.5 (unit 2.8)
  • Pepsin (2500 to 3500 U/mg; Sigma)
  • 2 M Tris base
  • 0.1 M cysteine
  • PBS ( appendix 2A)
  • 26 × 900–mm column with appropriate size‐exclusion (SE) resin ( appendix 3I)
  • Additional reagents and equipment for protein dialysis ( appendix 3H), SE chromatography ( appendix 3I), and SDS‐PAGE (unit 8.4)

Basic Protocol 2: Fragmentation of IgM to IgMs Subunit

  Materials
  • 3 to 5 mg/ml purified IgM antibody in PBS (unit 2.9)
  • 0.1 M cysteine stock solution (free base, crystalline; Sigma)
  • Borate‐buffered saline (unit 2.7) with 3 mM EDTA
  • 26 × 900–mm column with appropriate size‐exclusion (SE) resin ( appendix 3I)
  • Additional reagents and equipment for SE chromatography ( appendix 3I), protein dialysis ( appendix 3H), and SDS‐PAGE (unit 8.4)
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Figures

Videos

Literature Cited

Literature Cited
   Beale, D. and Van Dort, T. 1982. A comparison of the proteolytic fragmentation of IgM from several different mammalian species. Comp. Biochem. Physiol. (B)71:475‐482.
   Maillet, T., Roche, A.‐C., Therain, F., and Monsigny, M. 1985. Time course localization of IgM monoclonal antibody and its fragments in leukemic tumor‐bearing mice. Cancer Immunol. Immunother. 19:177‐182.
   Marks, R. and Bosma, M.J. 1985. Truncated µ (µ′) chains in murine IgM: Evidence that µ´ chains lack variable regions. J. Exp. Med. 162:1862‐1877.
   Metzger, H. 1970. Structure and properties of IgM. In Advances in Immunology, Vol. 12 (F.J. Dixon and H.G. Kunkel, eds.) pp. 57‐116. Academic Press, San Diego.
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