Separation of antigens and antibodies by immunoaffinity chromatography

Figures & data

Figure 1.  (a) Schematic representation of an IgG molecule. IgG has a basic four-chain monomeric structure consisting of two identical heavy chains which contains one variable domain (V_H) and three constant domains (C_H1, C_H2, and C_H3), and two identical light chains. Between the C_H1 and C_H2 is the hinge region. An IgG molecule can be divided into two parts functionally: Fragment antigen-binding (Fab) fragment, which is the antigen-binding site, and Fragment crystallizable (Fc) fragment, which is the protein A-binding site (Yang et al., 2003). (b) The Staphylococcal protein A shown here is comprised with five homologous IgG-binding domains (E, D, A–C), which have high affnity with the side chains of His435 and Tyr436 of Fc1, and a cell-wall attaching structure (XM) (Hober et al., 2007).

Table 1.  Examples of purification of antibodies using synthetic mimic ligands of proteins A and L.

Mimic ligand	Target antibody	Capacity	Purity (%)	References
Ligand 8/7	IgG, Fab	0.5 mg Ig/g	90–95	Roque et al. (2005a)
TG19318	IgG, IgM, IgE, IgY	10–60 mg Ig/g	90–95	Kabir (2002)
D-PAM	IgG, Polyclonal IgG	50–60 mg Ig/g	>90	D’Agostino et al. (2008)
Ligand 22/8	IgG, IgA, IgM	52 mg Ig/g	97–99	Kabir (2002)
MAbsorbent A1P/A2P	Polyconal IgG	27 mg IgG/ml	>95	Ghose et al. (2006)

Table 2.  Properties of the Ig-binding bacterial proteins A, G and L.

Antibodies		Protein A	Protein G	Protein L
Human Ig	Ig G1,2,4	+++	+++	+++
	IgG3	–	+++	+++
	IgM, IgA, IgE,IgD	+	+	++
	Fab, F(ab′)2	+	+	++
	scFv	+	–	+++
Mouse Ig	IgG1, IgG2	+	++	++
	IgG2a, IgG2b	++	++	++
	IgM	+	–	++
	IgA	++	+	++
Polyclonals	Mouse	++	++	++
	Rat	+	++	++
	Rabbit	++	+++	+
	Goat, bovine	+	++	–
	Porcine	+	+	++

+++ represents very strong binding; ++ represents strong binding; + represents moderate binding; – represents no binding.

Yang L, Biswas ME, Chen P. (2003). Study of binding between protein A and immunoglobulin G using a surface tension probe. Biophys J, 84, 509–522.

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Hober S, Nord K, Linhult M. (2007). Protein A chromatography for antibody purification. J Chromatogr B Analyt Technol Biomed Life Sci, 848, 40–47.

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Roque AC, Taipa MA, Lowe CR. (2005a). An artificial protein L for the purification of immunoglobulins and fab fragments by affinity chromatography. J Chromatogr A, 1064, 157–167.

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Kabir S. (2002). Immunoglobulin purification by affinity chromatography using protein A mimetic ligands prepared by combinatorial chemical synthesis. Immunol Invest, 31, 263–278.

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D’Agostino B, Bellofiore P, De Martino T, Punzo C, Rivieccio V, Verdoliva A. (2008). Affinity purification of IgG monoclonal antibodies using the D-PAM synthetic ligand: Chromatographic comparison with protein A and thermodynamic investigation of the D-PAM/IgG interaction. J Immunol Methods, 333, 126–138.

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Ghose S, Hubbard B, Cramer SM. (2006). Evaluation and comparison of alternatives to protein A chromatography Mimetic and hydrophobic charge induction chromatographic stationary phases. J Chromatogr A, 1122, 144–152.

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