Bio-Rad Affi-Gel Hz Hydrazide Gel User Manual
Affi-Gel®Hz
Immunoaffinity
Kit
Instruction
Manual
Catalog Number
153-6060
For Technical Service
Call Your Local Bio-Rad Office or
in the U.S. Call 1-800-4BIORAD
(1-800-424-6723)
Table of Contents
Section 1 Introduction...............................................................1
1.1 Immunoaffinity Coupling and Usefulness of the
Immobilized Antibody.........................................................1
1.2 Hydrazide Coupling Chemistry...........................................2
1.3 Diagram of Kit Components ...............................................2
Section 2 Monoclonal Antibodies.............................................3
Section 3 Antibody Purification...............................................3
Section 4 Immobilization Protocol...........................................4
4.1 Buffer Exchange..................................................................4
4.2 Oxidation of IgG..................................................................6
4.3 Desalting Procedure.............................................................7
4.4 Coupling of Oxidized IgG to Affi-Gel Hz
Hydrazide Gel......................................................................7
Section 5 Applications of Affi-Gel Hz Immobilized IgG........9
5.1 Conditioning the Immunoaffinity Column..........................9
5.2 Sample Application ...........................................................10
5.3 Elution Suggestions...........................................................10
Section 6 Product Information...............................................11
6.1 Specifications.....................................................................11
6.2 Ordering Information.........................................................11
Section 1
Introduction
The Affi-Gel Hz immunoaffinity kit is a unique approach to
IgG coupling to an agarose support matrix for affinity purif ication.
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 vicinal 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 binding site by allowing the correct orientation of the antibody.
1.1 Immunoaffinity Coupling and Usefulness
of the Immobilized Antibody
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 carbohydrate of the Fc region.
Carbohydrate moieties on Fc region of IgG.
Hydrazide functional group on gel spacer arm.
Immunoglobulin G.
1
vs
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.
1.2 Hydrazide Coupling Chemistry
HOH
HOH2C
OH OH
OH
IgG
NalO
4
C
IgG
2
Affi-Gel Hz
OH
O
O
gel
HOH2C
NH
Affi-Gel Hz
gel
NH
IgG
OH
NN
Sugar residue of
carbohydrate on the
Fc region of IgG.
Periodate oxidation
of vicinal hydroxyls
to form aldehydes.
Oxidized IgG specific
coupling to Affi-Gel
Hz hydrazide gel.
1.3 Diagram of Kit Components
Econo-Pac
Desalting Columns
1 x 10 cm
Econo-Column
Chromatography
Column
Affi-Gel Hz 10 x
Coupling Buffer, 25 ml
Affi-Gel Hz Oxidizer,
25 mg NalO
4
each
Materials and Equipment Not Included in this Kit
Affi-Gel Hz Hydrazide
Gel, 5 ml
®
10DG
®
Essential
15 ml screw cap test tubes - polypropylene or polystyrene, leak-free
End-over-end rotating mixer
Transfer pipets
pH meter
UV-visible spectrophotometer
2
Optional
Bio-Rad Protein Assay Kit I (catalog number 500-0001)
Section 2
Monoclonal Antibodies
Due to the unique specificity of monoclonal antibodies, coupling to Affi-Gel Hz gel may not be optimal for all monoclonal
antibodies. Loss of activity may occur depending on the individual
monoclonal. T o minimize loss of v aluable antibody preparations, BioRad highly recommends that a sample coupling experiment be performed to determine the efficiency of the coupling reaction. After
the results of the sample coupling are determined, the remaining
antibody can be coupled to the Affi-Gel Hz gel. If results are less than
adequate with the Affi-Gel Hz gel, use Af fi-Gel 10 gel to immobilize the monoclonal antibody.
Section 3
Antibody Purification
Prior to IgG coupling to Affi-Gel Hz hydrazide gel, the antibody must be at least partially purified. A high degree of purity will
insure that higher IgG concentrations are available for oxidation
and immobilization to the affinity support. This increased purity of
IgG will maximize coupling capacity and purification potential.
There are many approaches to antibody purification, and only a few
will be presented here.
For use with this kit, the purified antibody should be in a total
protein concentration of 1-5 mg/ml in a total maximum volume of
5 ml prior to buffer exchange. This recommended volume is necessary for rapid desalting and buffer exchange with the Econo-Pac
10DG columns. Regardless of the purification method(s) chosen, it
will be necessary to buffer exchange the purified antibody into the
Affi-Gel Hz coupling buffer prior to oxidation and coupling following the procedure in this manual. See Buffer Exchange, Section 4.1.
IgG from serum or ascites can be purified by using a combination of ion exchange with the Macro-Prep®high S support and
hydrophobic interaction chromatography with the Macro-Prep
t-butyl support (or a combination of affinity chromatography
with CM Affi-Gel blue gel and hydroxyapatite chromatography
with Macro-Prep CHT, type I support). DEAE Affi-Gel blue chro-
3
®
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