Immunoaffinity chromatography uses the specific binding between antigens and antibodies to purify proteins. Monoclonal antibodies are commonly used as they are highly specific and customizable. The antibodies are immobilized onto a matrix to purify target proteins from mixtures by retaining antigen-antibody complexes on the column while other molecules pass through. Various techniques are used to elute and collect the purified protein. Flow cytometry also utilizes fluorescent antibody binding to identify and separate cells based on surface markers allowing purification of cell populations.

1. Applications of Antibodies in purification
Presented by
Ankur Sharma
M.Sc Ist year (ABTC)

2. Use of highly specific interaction between
antigen & antibody to purify the protein
present in small quantities as a mixture with
several types of molecules is called
immunopurification.
Due to customized avidity and specificity,
monoclonal antibodies have become
indispensable tool for both protein
characterization and purification.

3. Immunoaffinity chromatography is a
process in which affinity of an antigen to
specific antibody is utilized as basis of
separation.
Affinity chromatography enables
purification of biomolecules on the basis of
their chemical structure or biological
function.

4. The method was invented in 1968 by Pedro
Cuatrecasas and Meir Wilchek.
The ligand ( here antibody) is immobilized
covalently to chromatographic matrix to yield an
active immunosorbent.
The use of antibodies as the immobilized ligand
has been exploited in isolation and purification
of a range of proteins including membrane
proteins of viral origin.

5. Mechanism of immunoaffintiy chromatography :

7. The Monoclonal antibodies are fixed to insoluble
matrix like dextran or agarose bead by cross linking
agents like cyanogen bromide, N-hydroxysuccinamide
(NHS),carbonyl di imidazole(CDI)
Another technique uses a solid support coated with an
antibody binding protein, such as Protein A or G,
which captures and immobilizes the antibodies.
The antibody is then covalent linked to the resin with
the aid of a chemical cross linker.
Procedure:-

8.  The bacterial proteins, protein A and protein
G are the most commonly used antibody
binding proteins.
 Protein A is a 42 kDa protein that is a normal
constituent of S. aureus bacteria cell walls.
 Protein G is a 30-35 kDa protein that is found
in the cell wall of β-hemolytic streptococci of
the C and G strains.

9.  The beads are packed in a column
through which the solution containing the
protein to be purified is passed.
 The interaction of Ag and Ab form Ag-Ab
complex which is retained in the column
while remaining molecules freely pass
through the column.
 Suitable washing procedure allow
collection of purified antigen held as Ag-
Ab complex in column.

10.  The reversible interaction between Ag
and Ab can be disrupted to yield a
highly purified product in the column
eluate.
 Elution of the bound protein quite often
requires forceful conditions because
very tight binding with antibody.
 Elution procedure include use of high
salt concentrations with or without

11.  detergent and the use of urea or
guanidine hydrochloride, both of which
cause denaturation of protein.
 To avoid denaturation some chaotropic
agents such as thiocynate, perchlorate
and trifluoroacetate are used.
 Another option is lowering the pH near
about 3

12. Immunoaffintiy chromatography

13. Purification of Human Chorionic
Gonedotropin (hCG) from pregnancy urine
by immunoaffinity chromatography using
a monoclonal antibody anti b chain hCG.
Identification of carcino embryonic
antigen(CEA) related Ag in mecmium and
colorectal carcinoma tissue by western
blotting and differential immunoaffinity
chromatography.

14. Immunoaffinity purification of factor IX
(Christmas factor) by using conformation-specific
antibodies directed against the factorIX-metal
complex:-
Factor IX is a vitamin K dependent zymogen that
is functionally defective or absent in patients with
hemophilia B.
A method of imnmunoaffinity chromatogiaphy has
been developed for a one-step high yield
purificationof factor IX directly fromplasma.

15. The technique utilizes conformation-specific
antibodies that bind solely to the metal-
stabilized factor IX conformer, but not to the
conformer of factor IX found in the absence
of metal ions
Active eukaryotic RNA polymerase II
(RNAP II) is purified by immunoaffinity
chromatography, using a monoclonal
antibody (mAb) that reacts with the highly
conserved hepta peptide repeat of the largest
subunit.

16. This MAb (designated 8WG16) is conjugated to
CNBr-activated Sepharose and used to purify
RNAPII from wheat germ and calf thymus.

17. INTRODUCTION
Flow cytometer was designed to automate
the analysis and separation of cells stained
with fluorescent antibody.
Flow cytometer which is also known as
Fluorescence activated cell sorter(FACS) is a
technique for counting, examining and
sorting microscopic objects suspended in a
fluid.
In 1944 Albert Coons gave the concept of
technique immunofluorescence

18. Principle:-
Flow cytometer is based on immuno
fluorescence.
According to it, antibodies could be
labeled with fluorescent molecules
called fluorochrome which absorb
light of one wavelength(excitation)
and emit light of another
wavelength(emission).
Most commonly used fluorochromes
are:

19. Fluorescein :-
absorbs blue light(490nm) and
emits intense yellow-green light(517nm).
Rhodamine :-
absorbs yellow-green(515nm) and emits
deep red(546nm)
Pycoerythrinh :-
it is efficient absorber of light(~30 fold
greater than fluorescein) and emits red light.

20. It may be more convenient to use a second
non specific fluorescent anti Ig to bind to the
unlabeled specific Ab bound to cell surface
Ag.
the antibody conjugate will bind to only those
cells that exibit the concerned Ag on their
surface
Such cells are therefore identified and
separated due to their fluorescence.

21. The flow cytometer uses a laser beam and
light detector to count single intact cell in
suspension.
The mixture containing cells to be purified
is loaded in the sample chamber of the
flow cytometer.
The cells are expelled, one at a time, from
the vibrating nozzle that generates micro
droplets.

22. As it leaves the nozzle each droplet receive
small electric charge and attached computer
can detect the exact time at which a particular
droplet will pass through the deflection plates
By applying a momentary charge to the
deflection plates when a droplet is passing
between them it is possible to deflect the path
of a particular droplet into one or another
collecting vessels.
This allows sorting of population of cells into
subpopulations having different profile of
surface markers.

23. Flow cytometer
Kuby IMMUNOLOGY SIXTH EDITION

24. DNA Measurement and Cell Cycle Analysis
by Flow Cytometry.
Two of the most popular flow cytometric
applications are the measurement of cellular
DNA content and the analysis of the cell cycle.
The nuclear DNA content of a cell can be
quantitatively measured at high speed by flow
cytometry .
Initially, a fluorescent dye that binds
stoichiometrically to the DNA is added to a
suspension of permeabilized single cells.
.

25. The principle is that the stained material has
incorporated an amount of dye proportional to the
amount of DNA.
The stained material is then measured in the flow
cytometer and the emitted fluorescent signal
yields an electronic pulse with a height (amplitude)
proportional to the total fluorescence emission from
the cell.
Thereafter , such fluorescence data are considered a
measurement of the cellular DNA content.

26. Flow cytometry assay for intracellular rabies virus
detection
A suspensions of BHK-21 and C6 cells were infected
with fixed rabies virus strain (Pasteur Virus
strain—PV) or a wild rabies virus strain (WRS) and
a time course of virus infection was established.
Infected and control uninfected cells were fixed,
permeabilized, and stained with a rabies virus-
specific antibody conjugate.
This was achieved byre suspending cells in a
solution containing p-formaldehyde in FACS lysis
fluid, which allowed the detection of intracellular
virus with flourescein-coupled antibodies by flow
cytometry