Bio-Rad Affi-Gel Hz Hydrazide Gel - Manual (Page 3)

Section 1
Introduction
The Affi-Gel Hz immunoaffinity kit is a unique approach to
IgG coupling to an agarose support matrix for affinity purification.
This kit achieves a more uniform orientation of coupled antibody than
currently available activated supports which couple via primary
amines. Affi-Gel Hz hydrazide gel is an agarose support which
reacts with the aldehydes of oxidized carbohydrates to form stable,
covalent hydrazone bonds. Immunoglobulin G is a glycoprotein
which contains approximately 3% carbohydrate localized on the
Fc region (heavy chain) of the antibody. Periodate oxidation of vic-
inal hydroxyls of the sugars of these carbohydrates forms aldehyde
groups for specific coupling to Affi-Gel Hz gel. This coupling
through the carbohydrate eliminates the loss of antibody activity
experienced in primary amino coupling at or near the antigen bind-
ing site by allowing the correct orientation of the antibody.
1.1 Immunoaffinity Coupling and Usefulness
of the Immobilized Antibody
1
Oriented coupling results in higher
antibody activity for greater antigen
binding capacity per coupled IgG.
IgG immobilized on Affi-Gel HZ
hydrazide gel through the carbohy-
drate of the Fc region. Random coupling yields low antigen
binding due to attachment at or near
the binding side of the antibody. IgG
coupling directly to agarose support
via primary amine.
Carbohydrate moieties on Fc region of IgG.
Hydrazide functional group on gel spacer arm.
Immunoglobulin G.
vs
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