Isolasi dan Pemurnian

Antibodi
Meiriza Djohari
Mata Kuliah Immunologi
STIFAR

Structure of Antibody

Fragmen antigen Binding

Fragmen crystallizable

What is Antibody Purification?

Antibody purification is a multi process by which
antibodies with high purity can be achieved--> for
immunochemical techniques within general research
and for therapeutic and diagnostic applications

Source of Antibody
Serum (polyclonal antibodies),
Ascites fluid
Cell culture supernatant of a hybridoma cell line
(monoclonal antibodies).

Why to Purifiy Antibody

To remove possible contaminants serum
protein such as albumin, transferrins, cell
degradation products like DNA and cellular
proteins

Purification Methods
The choice of a purification method based on these
factors :
Nature of antibody
Nature of feed stock
Scale of Production
Economics-- cost and other factors
Process timing
Desired purifity

Antibody Purification Process

Sample preparation
Capture
Initial purification
Secondary Purification
Polishing/Formulation

Antibody Purification
Step 1 : Sample Preparation
It is the initial step in which crude protein sample is
conditioned or making it ready for the initial capture
step
Involves changing Ph or Ionic strength, dilution of the
crude sample or addition of salts for the ionic
changing (cost ) --> Buffer exchange by size
exclution chromatography or to use ultrafiltration or
diafiltration

Antibody Purification
Step 2 : Capture

Physicochemical fractionation
Class-specific affinity
Antigen-specific affinity

Antibody Purification
Step 3 : Intermediate Purification
On the nature and the optimization
requirement of the crude antibody source.
In addition to protein contaminants, other
impurities such as DNA, Endotoxins,
viruses, and aggregate need to be
removed. In such case multistep
procedure are inevitable.

Antibody Purification
Step 4 : Polishing/Formulation
Final polishing/formulation step can be
considered as a part of purification in
which it removes condition that would
impair the stability or utility of the
antibody in its intended use.
Ultrafiltration
SEC, Dialfiltration
Lyophilization

Antibodies that were developed as

monoclonal antibody can be fully purified
without using an antigen-specific affinity
method because the target antibody is (for
most practical purposes) the only
immunoglobulin in the production sample.
By contrast, for polyclonal antibodies (serum
samples), antigen-specific affinity purification
is required to prevent co-purification of
nonspecific immunoglobulins.

Physicochemical fractionation

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation
Ion Exchange Chromatography
Immobilized Metal Chelate Chromatography
Thiophilic Adsorption
Melon Gel Chromatography

Differential precipitation, size-exclusion or solidphase binding of immunoglobulins based on :

Size
Charge
Other shared chemical characteristics of
antibodies in typical samples.

This isolates a subset of sample proteins that includes the immunoglobulins.

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation
Ion Exchange Chromatography
Immobilized Metal Chelate Chromatography
Thiophilic Adsorption
Melon Gel Chromatography

Dialysis, desalting and

diafiltration can be used
to exchange antibodies
into particular buffers and
remove undesired lowmolecular weight (MW)
components.
Dialysis membranes, sizeexclusion resins, and
diafiltration devices that
feature high-molecular
weight cut-offs (MWCO)
--> can be used to
separate immunoglobulins
(>140kDa) from small
proteins and peptides.

However, except with

specialized columns and
equipment, these
techniques alone cannot
purify antibodies from
other proteins and
macromolecules that are
present in typical antibody
samples.
More commonly, gel
filtration and dialysis are
used following other
purification steps, such as
ammonium sulfate
precipitation

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation
Ion Exchange Chromatography
Immobilized Metal Chelate Chromatography
Thiophilic Adsorption
Melon Gel Chromatography

Ammonium sulfate precipitation is frequently used

to enrich and concentrate antibodies from serum,
ascites fluid or cell culture supernatant.
As the concentration of this lyotropic salt is
increased in a sample, proteins and other
macromolecules become progressively less soluble
until they precipitate; the lyotropic effect is called
"salting out."
Antibodies precipitate at lower concentrations of
ammonium sulfate than most other proteins and
components of serum.

The selectivity, yield, purity and reproducibility

of precipitation depends on :

Time
Temperature
PH
Rate of salt addition .

Ammonium sulfate precipitation

provides sufficient purification for some
antibody applications, but most often it
is performed as a preliminary step before
column chromatography or other
purification method

Other antibody precipitation reagents

that have been used for special antibody
purification situations include using
caprylic/octonoic acid, polyethylene
glycol and ethacridine .

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation
Ion Exchange Chromatography
Immobilized Metal Chelate Chromatography
Thiophilic Adsorption
Melon Gel Chromatography

Uses positively or negatively charged

resins to bind proteins based on their net
charges in a given buffer system (pH).
IEC is a cost-effective, gentle and reliable
method for antibody purification.

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation
Ion Exchange Chromatography
Immobilized Metal Chelate Chromatography
Thiophilic Adsorption
Melon Gel Chromatography

Uses chelate-immobilized divalent metal

ions (usually nickel, Ni2+) to bind
proteins or peptides that contain clusters
of three or more consecutive histidine
residues.

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation
Ion Exchange Chromatography
Immobilized Metal Chelate Chromatography
Thiophilic Adsorption
Melon Gel Chromatography

Combines the properties of hydrophobic

interaction chromatography (HIC) and
ammonium sulfate precipitation (the
lyotropic effect).

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation
Ion Exchange Chromatography
Immobilized Metal Chelate Chromatography
Thiophilic Adsorption
Melon Gel Chromatography

A proprietary resin chemistry (and optimized

buffer system) for purifying antibodies by
chemical-based fractionation.
In the specified mild buffer condition, Melon
Gel resin binds most non-IgG proteins found
in serum, ascites fluid and culture
supernatants, while allowing purified IgG to
be collected in the flow-through fraction.

Class-specific affinity

Solid-phase binding of particular

antibody classes (e.g., IgG) by
immobilized biological ligands (proteins,
lectins, etc.) that have specific affinity to
immunoglobulins.
This purifies all antibodies of the target
class without regard to antigen
specificity.

Protein A, G and L Antibody-binding Ligands

Antibody Purification with Protein A, G and L
IgM Purification
IgA Purification

Antigen-specific affinity

Purification of antigen-specific antibodies is

often required.
Can be accomplished by immobilizing the
particular antigen used for immunization so
that only those antibodies that bind
specifically to the antigen are purified in the
procedure.

Antigen Immobilization and Presentation

Peptide Antigens and Affinity Ligands
Protein Antigens and Affinity Ligands
Binding and Elution Conditions

Tugas
Cari jurnal tentang purifikasi antibodi
Dibagi 6 kelompok
klp 1 : Size Exclusion Chromatography
klp 2 :Ammonium Sulfate Precipitation
Klp 3 :Ion Exchange Chromatography
Klp 4 :Immobilized Metal Chelate
Chromatography
Klp 5 :Thiophilic Adsorption
Klp 6 :Melon Gel Chromatography

Terima Kasih