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Purification of Human IgA

Todd D. Pack¹

¹Applied 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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Unit Introduction
Basic Protocol: 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
Bibliography

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Materials

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

Materials

Immobilized IgA-bp (Athens Research and Technology)
Carbonate elution and wash buffers (see recipe)
Human plasma, colostrum, serum, milk, or saliva
TBST-MgCl₂ wash buffer (optional; see recipe)
MgCl₂ 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

Additional Materials (also see Basic Protocol)

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

Additional Materials (also see Basic Protocol)

Ammonium sulfate
DEAE column buffer (see recipe)
DEAE-Sepharose (Pharmacia Biotech)
DEAE column buffer containing 0.10 M and 0.25 M NaCl
TBS-NaN₃ (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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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.
	Lamm, M.E. 1997. Interactions of antigens and antibodies at mucosal surfaces. Annu. Rev. Microbiol. 51:311-340.
	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.
These three papers above are excellent in-depth reviews of the structure and function of human IgA, covering biosynthesis to antigen interaction.
	Mestecky and Kilian, 1985. See above.
Lists and reviews fractionation procedures for IgA purification and cites numerous corresponding references.