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Isolation of Murine and Human Immunoglobulin M and Murine Immunoglobulin D

Sarah M. Andrew1,  Julie A. Titus2,  Ashok Amin3,  Richard Coico4

1Lancaster University, Lancaster, United Kingdom
2National Cancer Institute, Bethesda, Maryland
3Carilion Clinic, Roanoke, Virginia
4Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania




Publication Name: 
Current Protocols in Immunology
Unit Number: 
UNIT 2.9
DOI: 
10.1002/0471142735.im0209s85
Online Posting Date: 
April, 2009
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Abstract

This unit describes two classical protocols for the purification of IgM—dialysis of ascites fluid, tissue culture medium, or bioreactor supernatants against distilled water to precipitate pure IgM, and ammonium sulfate precipitation. Both protocols can be followed by size-exclusion chromatography to obtain a highly purified product. Recently, an affinity method for purification of IgM has been developed using mannan-binding protein, and is described here. The third approach presented is a one-step IgD purification method, designed specifically for murine derived samples, that uses Sepharose coupled to lectin derived from the seeds of Griffonia simplicifolia-1. This represents a simple, rapid, and gentle, approach to isolating this highly labile immunoglobulin from IgD-containing ascites or hybridoma sources. Curr. Protoc. Immunol. 85:2.9.1-2.9.8. © 2009 by John Wiley & Sons, Inc.

Keywords: IgM; IgD; purification; murine; human

     
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Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of IgM by Dialysis and Size-Exclusion Chromatography
  • Alternate Protocol 1: Purification of IgM by Ammonium Sulfate Precipitation
  • Alternate Protocol 2: Affinity Purification with Mannan-Binding Protein
  • Basic Protocol 2: Purification of Murine IgD by Lectin Affinity Chromatography
  • Commentary
  • Literature Cited
  • Other Versions
     
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Materials

Basic Protocol 1: Purification of IgM by Dialysis and Size-Exclusion Chromatography

 Materials
  • Ascites fluid or monoclonal antibody (MAb) supernatant (unit 2.6)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • Sorvall centrifuge and SS-34 rotor (or equivalent)
  • Dialysis tubing (appendix 3H)
  • 26 × 900–mm column with appropriate size-exclusion (SE) resin (appendix 3I)
  • Additional reagents and equipment for purification of immunoglobulin G (unit 2.7), protein dialysis (appendix 3H), size-exclusion chromatography (appendix 3I), and SDS-PAGE (unit 8.4)

Alternate Protocol 1: Purification of IgM by Ammonium Sulfate Precipitation

 Additional Materials (also see Basic Protocol 1)
  • Saturated ammonium sulfate (SAS; unit 2.7)
  • Ammonium sulfate crystals (CAS)
  • Additional reagents and equipment for purification of IgG (unit 2.7) and size-exclusion chromatography (appendix 3I)

Alternate Protocol 2: Affinity Purification with Mannan-Binding Protein

 Additional Materials (also see Basic Protocol 1)
  • EDTA (ethylenediaminetetraacetic acid disodium salt)
  • Ascites fluid or monoclonal antibody (MAb) supernatant (unit 2.6)
  • NaOH
  • Ultralink immobilized mannan-binding protein (MBP, Pierce)
  • Binding buffer: 0.1 M NaHCO3/0.5 M NaCl, pH 8.3, 4°C and room temperature
  • 1.0 M potassium iodide
  • Phosphate-buffered saline (PBS; appendix 2A)
  • 1.5 × 10–cm column or 50-ml centrifuge tube
  • MSE Mistral 3000i refrigerated centrifuge (or equivalent)
  • Additional reagents and equipment for dialysis (appendix 3H) and column chromatography (appendix 3I), SDS-PAGE (unit 8.4), and ELISA (unit 2.1)

Basic Protocol 2: Purification of Murine IgD by Lectin Affinity Chromatography

 Materials
  • Mice bearing IgD plasmacytomas (e.g., TEPC-1017), IgD-secreting hybridoma (e.g., B-18), or MAb-producing culture
  • PBS-CA: PBS (appendix 2A) containing 1 mM CaCl2, pH 7.0 (ice cold)
  • PBS-CA-GAL: PBS-CA containing 0.1 M d-galactose, pH 7.0 (ice cold)
  • Delipidating agent (Beckman)
  • GS-I lectin–Sepharose (E-Y Labs)
  • PBS, pH 7.3 (appendix 2A), ice cold
  • 0.45-µl disc-cartridge filtration membrane (e.g., Amicon)
  • Sorvall centrifuge and SS-34 rotor or equivalent
  • Additional reagents and equipment for producing ascites fluid (unit 2.6), protein dialysis (appendix 3H), column chromatography (appendix 3I), and reducing SDS-PAGE (unit 8.4)
Looking for materials?  Suppliers Guide
     
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Literature Cited

Literature Cited
    Argon, Y. and Milstein, C. 1984. Intracellular processing of membrane and secreted immunoglobulin 100 chains. J. Immunol. 133:1627-1634.
    Hallström, T., Muller, S.A., Morgelin, M., Mollenkvist, A., Forsgren, A., and Riesbeck, K. 2008. The Moraxella IgD-binding protein MID/Hag is an oligomeric autotransporter. Microbes. Infect. 10:374-381.
    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, Boston.
    Murphy, L.A. and Goldstein, J. 1977. Five -d-galactopyranosyl-binding isolectins from Bandieraea simplicifolia seeds. J. Biol. Chem. 252:4739-4742.
    Nethary, A., Raison, R.L., and Esterbrook-Smith, S.D. 1990. Single-step purification of immunoglobulin M on C1q-Sepharose. J. Immunol. Methods 126:57-60.
    Ohta, M., Okad, M., Yamashina, I., and Kawasaki, T. 1990. The mechanism of carbohydrate-mediated complement activation by the neuraminidase-binding protein. J. Biol. Chem. 265:1980-1984.
    Oppenheim, J.D., Amin, A.R., and Thorbecke, G.J. 1990. A rapid one-step purification procedure for murine IgD based on the specific affinity of Bandieraea (Griffonia) simplicifolia-1 for N-linked carbohydrates on IgD. J. Immunol. Methods 130:243-250.
    Samuelsson, M., Hallstron, T, Forsgren, A., and Riesbeck, K. 2007. Characterization of IgD-binding site of Haemophilus influenza serotype b. J. Immunol. 178:6316-6319.
    Vasilov, R.G. and Ploegh, H.L. 1982. Biosynthesis of murine immunoglobulin D: Heterogeneity of glycosylation. Eur. J. Immunol. 12:804-813.
     
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