Immobilized metal-ion affinity chromatography of human antibodies and their proteolytic fragments.
J Chromatogr B Analyt Technol Biomed Life Sci
; 808(1): 57-62, 2004 Aug 25.
Article
em En
| MEDLINE
| ID: mdl-15236686
ABSTRACT
Immobilized metal-ion affinity chromatography (IMAC) performed with four different transition metal ions copper(II), nickel(II), zinc(II) and cobalt(II), was used to study the adsorption properties of human polyclonal gamma-globulines (IgG), Cohn II-III fractions, and their pepsin cleaved fragments Fab'2 and F'c. In each case, digested products showed lower affinity for metal ions, as well by decreasing pH elution as by competition with imidazole. An explanation was proposed by the presence of a histidine (His) cluster in the F'c domain of IgGs, identified by computer calculation (accessible surface area (ASA) determination) as the more probable His 433-x-His 435 sequence presented in the CH3 domain of human IgG heavy chain. As shown by IMAC and electrophoresis, F'c and undigested IgG have higher affinity for transition metal ions than Fab'2 fragments and could be then separated in one step by IMAC. When chelated Zn(II) or Co(II) are used as ligands, the Fab'2 fragment could be easily recovered under mild conditions (pH 7) in the non-retained fraction. This approach could be used as a powerful alternative to conventional protein A/G methods for the commercial preparation of non immunogen active Fab'2 fragments.
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Base de dados:
MEDLINE
Assunto principal:
Fragmentos de Imunoglobulinas
/
Cromatografia de Afinidade
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Metais
/
Anticorpos
Idioma:
En
Ano de publicação:
2004
Tipo de documento:
Article