Purification of Human IgA

Todd D. Pack1

1 Applied Biotech, San Diego, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 2.10B
DOI:  10.1002/0471142735.im0210bs38
Online Posting Date:  May, 2001
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Abstract

This unit describes the purification of human immunoglobulin A (IgA).The main method utilizes an IgA‐binding protein (IgA‐bp) as an affinity reagent, similar to the IgG‐binding proteins, protein A and protein G. An alternate protocol describes a method for isolating IgA1 using affinity column chromatography with Jacalin, an IgA1‐specific lectin. Another alternate protocol describes IgA purification by conventional fractionation using size‐exclusion chromatography.

     
 
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Table of Contents

  • Basic Protocol 1: Affinity Purification of Human IgA Using an IgA‐Binding Protein
  • Alternate Protocol 1: Affinity Purification of Human IgA1 Subclass Using Jacalin
  • Alternate Protocol 2: Purification by Conventional Fractionation
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Affinity Purification of Human IgA Using an IgA‐Binding Protein

  Materials
  • Immobilized IgA‐bp (Athens Research and Technology)
  • recipeCarbonate elution and wash buffers (see recipe)
  • Human plasma, colostrum, serum, milk, or saliva
  • recipeTBST‐MgCl 2 wash buffer (optional; see recipe)
  • recipeMgCl 2 elution buffer (optional; see recipe)
  • 10 × 1.5‐cm plastic or glass chromatography column
  • Sorvall RC series centrifuge and SS‐34 rotor (or equivalent)
  • Automated liquid chromatography system (optional)
  • Additional reagents and equipment for column chromatography ( appendix 3I)

Alternate Protocol 1: Affinity Purification of Human IgA1 Subclass Using Jacalin

  • Immobilized Jacalin (Vector Laboratories)
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A)
  • 0.8 M galactose in PBS, pH 7.4
  • 10‐12k MWCO dialysis tubing
  • Additional reagents and equipment for protein dialysis ( appendix 3H)

Alternate Protocol 2: Purification by Conventional Fractionation

  • Ammonium sulfate
  • recipeDEAE column buffer (see recipe)
  • DEAE‐Sepharose (Pharmacia Biotech)
  • recipeDEAE column buffer containing 0.10 M and 0.25 M NaCl
  • recipeTBS‐NaN 3 (see recipe)
  • 1.6 × 70‐cm Sephadex G‐200 column (Pharmacia Biotech) or equivalent
  • Gradient former (optional)
  • 10‐12k MWCO dialysis tubing
  • Additional reagents and equipment for dialysis and concentration of samples ( appendix 3H), SDS‐PAGE (unit 8.4), immunostaining (unit 8.10), and removal of IgG contamination (unit 2.7)
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Figures

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Literature Cited

Literature Cited
   Mestecky, J. and Kilian, M. 1985. Immunoglobulin A (IgA). Methods Enzymol. 116:37‐75.
   Pack, T.D. 1999. Bacterial binding protein for single‐step purification of human IgA. Am. Biotechnol. Lab. 17:16‐18.
   Roque‐Barreirra, M.C. and Campos‐Neto, A. 1985. Jacalin, an IgA‐binding lectin. J. Immunol. 134:1740‐1743.
Key References
   Kerr, M.A. 1990. The structure and function of IgA Biochem. J. 271:285‐296.
  These three papers above are excellent in‐depth reviews of the structure and function of human IgA, covering biosynthesis to antigen interaction.
   Lamm, M.E. 1997. Interactions of antigens and antibodies at mucosal surfaces. Annu. Rev. Microbiol. 51:311‐340.
  Lists and reviews fractionation procedures for IgA purification and cites numerous corresponding references.
   Mestecky, J. and McGhee, J.R. 1987. Immunologlobulin A (IgA): Molecular and cellular interactions involved in IgA biosynthesis and immune response. Adv. Immunol. 40:153‐245.
   Mestecky and Kilian, 1985. See above.
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