Affinity chromatographic purification of immunoglobulin M antibodies utilizing immobilized mannan binding protein

doi:10.1016/0021-9673(92)80117-D

Journal of Chromatography A

Volume 597, Issues 1–2, 24 April 1992, Pages 247-256

https://doi.org/10.1016/0021-9673(92)80117-D Get rights and content

Abstract

A method is described for the rapid and efficient affinity chromatographic purification of murine monoclonal immunoglobulin M (IgM) which utilizes immobilized rabbit mannan binding protein (MBP). This solid-phase matrix is shown to bind IgM-class antibodies from a variety of species. Conditions reported show a binding capacity of IgM from murine ascites of nearly 1 mg/ml of immobilized MBP support. The prepared gel is shown to possess an ability to bind not only mouse IgM, but also human and bovine IgM, although with a lesser affinity. The matrix can be regenerated and reused at least ten times without any apparent loss of binding capacity or specificity. Mouse monoclonal IgM purified from ascites fluid using this method is greater than 95% pure as shown by high-performance liquid chromatography analysis.

References (15)

H. Hjelm et al.
FEBS Lett.
(1972)
M. Eliasson et al.
J. Biol. Chem.
(1988)
H.F. Deutsch et al.
J. Biol. Chem.
(1958)
A. Nethery et al.
J. Immunol. Methods
(1990)
M. Ohta et al.
J. Biol. Chem.
(1990)
Y. Kozutsumi et al.
Biochem. Biophys. Res. Commun.
(1980)
P. Cuatrecasas
J. Biol. Chem.
(1970)

There are more references available in the full text version of this article.

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Affinity chromatographic purification of immunoglobulin M antibodies utilizing immobilized mannan binding protein

Abstract

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Immunol. Methods

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.