UNIT III

• Isolation of lymphoid cells

• Antigen-Antibody Interactions
• Antigen-Antibody based experiments
•Qualitative Assays
•Hemagglutination,
•Precipitation
•Various Diffusion methods
•Electrophoresis and Western Blot
•Immunocytochemistry
•Quantitative Assays
•Radioimmunoassay
•Enzyme Immunoassay (ELISA)
•Flow cytometry

• Cell culture and experimental models

• Analysis of gene expression
 Isolation of lymphoid cells
• Human beings: Peripheral blood mononuclear cells (PBMC)
isolated from venous blood
• Animals: Bone marrow
Thymus
PBMC in blood
Spleen
Lymph nodes
Lymphoid tissues?
Stem cells from bone marrow and T lymphocytes from thymus used
to estimate their numbers.
Immune cells from peripheral blood, spleen, lymph nodes, and
lymphoid tissues are used to investigate the ability of these cells to
respond to antigens
• Isolation of organs and blood, and subsequent processing for
isolation of lymphoid cells are done in sterile (aseptic) manner
• Lymphoid organs are processed by homogenizing (stomacher- a
gentle homogenizer) and cells are isolated after repeated washing
in appropriate media

۞ Separation of cells by
density can be performed
by centrifugation at low g
with conventional
centrifuge.

۞ Serum albumin, dextran,

Ficoll, metrizamide, and
Percoll have all been used
successfully to separate
cells.
Figure A-23
• Immune cells do not respond until exposed to antigens.
• Hence, cells are treated in vitro with antigens or mitogens to
investigate the immune status of the animals/human beings
– E.g., of mitogen and antigen: Phytohemagglutinin, concanavalin A,
pokeweed mitogen, Candida antigen, and tetanus antigen

• Immune status is examined through the ability of immune cells

through qualitative and quantitativemethods
• Qualitative Methods:
– Western blot (protein estimation)
– Immunocytochemistry/immunofluorescence (cells in tissues)

• Quantitative Methods:
– Kill cells (NK cell activity using YAC-1 cells incubated with Cr51;
Radioimmunoassay)
– Produce cytokines/antibodies (ELISA technique)
– Proliferation (Tritiated thymidine incorporation)
– Cell number (Flow cytometry)
Antigen-Antibody Interactions
 Reactions between
antibody or antibody
fragments and antigen or
haptens

• Cross-linking of various
antigens by antibody is
required for precipitation,
agglutination, or complement
activiation
• It is only possible if the
antigen is multivalent and the
antibody is divalent either
intact or F(ab’)2.
• No cross-linking is
possible if the antigen or
antibody is univalent
 100 kDa

150 kDa

→ numerous small peptides

Fab fragment:
antigen binding
45 kDa

Fc fragment: 22 kDa
crystallizable
50 kDa

50-55 kDa
- Four types of non-covalent forces operates over a very short distance
( generally 1 angstrom )
- The interaction depends on a very close fit between the Ab & Ag.
Characterization and purification of
antibodies
• Detection of antigen/antibody reactions
difficult
• Sensitization is the binding of a specific
antibody to its specific antigen
• Can not be visualized
• Multitude of laboratory methods have been
developed to make this visible
 Three Distinct Phases of
Antigen/Antibody Reactions
• Primary Phenomenon – Sensitization
• Secondary Phenomenon – Lattice
formation
• Tertiary Phenomenon – Detected by effect
on tissues or cells.
 Primary phenomenon
• Sensitization – binding of antibody to antigen
– not visible
 Secondary Phenomenon
• Lattice Formation
• The Fab portion of the Ig molecule attaches to antigens
on to adjacent cells-visible results in agglutination
• If both antigen and antibody are SOLUBLE, reaction will
become visible over time, i.e., precipitation
 Tertiary Phenomenon
• Reaction not visible, detected by effect of
reaction on tissues or cells.
 Secondary Phenomena Most
Frequently Utilized
• Precipitation – soluble antibody reacts with
soluble antigen
• Agglutination – particulate antigens bound
together by antibody
• Complement Fixation – antibody binding to
antigen triggers activation of complement
 Strength of Ag-Ab Interactions

Antibody affinity
- is a quantitative measure of binding strength
- combined strength of the noncovalent interactions
between a binding site on an Ab & monovalent Ag

Antibody avidity
- Incorporates affinity of multiple binding sites
- True strength of the Ab-Ag interaction within
biological systems
- The interaction at one site will increase the possibility
of reaction at a second site
- High avidity can compensate for low affinity
( secreted pentameric IgM has a higher avidity than IgG )
 Affinity
• Antibody affinity is the strength of the
reaction between a single antigenic
determinant and a single combining site
on the antibody.

• It is the sum of the attractive and repulsive

forces operating between the antigenic
determinant and the combining site .
 Affinity
Affinity is the equilibrium constant that
describes the Ag-Ab reaction as illustrated in
the Figure. Most antibodies have a high affinity
for their antigens.
 Avidity
• Avidity is a measure of the overall strength of binding
of an antigen with many antigenic determinants and
multivalent antibodies.
• Affinity refers to the strength of binding between a
single antigenic determinant and an individual
antibody combining site whereas avidity refers to the
overall strength of binding between multivalent
antigens and antibodies.
• Avidity is influenced by both the valence of the
antibody and the valence of the antigen.
• Avidity is more than the sum of the individual
affinities.
Avidity
 Antigen-Antibody based
experiments: Qualitative Assays
 Hemagglutination
The antibodies are
induced by exposure
to cross-reacting
microbial antigens
present on common
intestine bacteria.
- ABO blood-group
antigens have subtle
differences in the
terminal residues of
the sugars on glyco-
proteins in RBC.
- - Providing the basis
for blood typing test
in blood transfusion

The highest dilution of serum that still causes agglutination but beyond which no
agglutination occurs is called titer
Agglutination does not occur high concentration of antibody. The tubes with high
concentration of serum where agglutination does not occur represent prozone.
Therefore, antiserum be tested for several dilutions.
 The Coombs test
• The surface of certain particulate antigens possess an electrical charge. E.g.
Net negative charge on the surface of RBCs by the presence of sialic acid.
• When such particles are suspended in saline, an electric potential called zeta
potential is created which prevents them from coming close to each other.
• This prevents IgG from agglutinating RBCs because the distance between
Fab arms of IgG molecule is too short. This causes repulsion due to zeta
potential.
• In contrast, Fab areas of IgM pentamers are far enough apart to bridge the
RBCs separated by zeta potential.
• However, Coombs (1950s) overcame this problem by developing a
technique to facilitated IgG to agglutinate erythrocyte antigens

• It is based on two important facts:

– 1) the immunoglobulins of one species (human) are immunogenic when injected into
another species (rabbit) and results in production of antibodies against the
immunoglobulins (in this case against human)
– 2) that many of the anti-immunoglobulins (rabbit anti-human Ig) will bind to Fc portion
of the antibody and allow Fab portion to react with antigen
The anti-immunoglobulin (Coombs test)

Direct Coombs test measures bound antibody

Indirect Coombs test measures serum antibody

Test is used for RBC typing in blood bank, diagnosis of

hemolytic diseases, tests for rheumatoid factor,
confirmatory test for syphilis, and the latex test for
pregnancy (detection of hCG in the urine of pregnant
women).
 Precipitation
• This describes the reaction between soluble antibody and
soluble antigen in which an insoluble product results.
• Please note the discussion describing the effects of antibody
excess, antigen excess, and the zone of optimal proportions
(equivalence zone) on the production of a precipitate.
• Remember that precipitation is a secondary phenomenon. Ag-
Ab reactions may occur and form soluble immune complexes
even without the production of a visible precipitate!

• Precipitation reactions can be done in a variety of ways: 

• In test tubes
• In agarose gels:
– As double diffusion
– As single diffusion
– After electrophoresis
 The Precipitin reaction

Precipitation of antigen-antibody complexes occurs because of the divalent antibody

molecules cross-link multivalent antigen molecules to form lattice.
 Passive Immunodiffusion
• Reactions in semisolid media such as agar gels
• Migrate towards each other and where they meet in optimal
proportions to form a precipitate.
• When soluble antigen and antibodies are placed in wells cut in
a gel (Fig 5.4 A), the reactants diffuse in the gel and form
gradients of concentration, with the highest concentrations
closest to the wells.
• If the antibody well contains antibodies 1, 2, and 3 specific for
antigens 1, 2, and 3 and diffuse at different rates, then 3 distinct
precipitin lines will form.
Ouchterlony Gel Diffusion (Double diffusion method)

• Holes punched in agar.

• Known antibody or antigen added to
center well.
• Known sample added to outer well.
• Unknown sample added to outer well next
to unknown sample.
• Wait for bands to form.
 Double-gel diffusions patterns

• A= pattern of identity
The precipitation appears as a continuous line in the form of an angle between those two
wells and the C well. There are no spurs at the angle and this type of reaction is termed
a band of identity

• B= pattern of non-identity
• C= pattern of partial identity (spur formation because Ab1 is different from Ag2)
Ouchterlony Immunodiffusion
 Radial diffusion
• A variation of double-diffusion test.
• The wells contain antigen at different concentrations and the
antibodies are distributed uniformly in the agar gel.
• Here, precipitin ring is formed.
• Used to measure serum proteins
 Radial Immunodiffusion

• A, B, C, D, and E represent known concentrations of antigen

• F and G represent unknown concentrations and can be determined
from the graph
 Electrophoretic Techniques
• Immunodiffusion can be combined with electrical current to speed
things up.
• Separating a mixture of proteins in an electrical field followed by
their detection with antibodies diffusing into the gel.
• Serum is placed in a small well cut in the center of a slide that is
coated with agar gel.
• Serum is subjected to electrophoresis, which separates the various
components according to their mobilities in the electrical field.
• After electrophoresis, a trough is cut along the side of the slides, and
antibodies to human serum proteins are placed in the trough.
• Precipitin lines form in the agar when the antibodies diffuse in the
agar and binds to the antigen at an optimal ratio
• Through this technique, the first antibody deficiency syndrome was
identified in 1952 (Bruton’s agammaglobulinemia)
Immunoelectrophoresis
 Rocket Immunoelectrophoresis

• Antigen is electrophoresis into gel containing

antibody. The distance from the starting well to the
front of the rocket shaped arc is related to antigen
concentration.
 Blotting
• The transfer of biomolecules onto a membrane support

1) Preparing a cell-free extract containing the biomolecules (antigens) of interest

• 2) Resolve the mixture by gel electrophoresis
• 3) Transfer the gel onto a protein-binding sheets (e.g., nitrocellulose) by electroblotting method
• 4) Incubate the membrane with a detection system (antibody) that specifically binds to the
molecule of interest
• 5) The antibody may be labeled (e.g. radioactivity) or a labeled anti-immunoglobulin to localized
the antigen and antibody complex.

Southerns - Detection of DNA fragments on a blot

Northerns - Detection of RNA fragments on a blot
Westerns - Detection of proteins on a blot
 Western Blots
• 1. What is Western blot?
A technique for detecting specific proteins separated by electrophoresis
by use of labeled antibodies. So called since it has some similarity to a
Southern blot.
• 2. Why we have to use it?
We can use this technique to identify a target protein in a complex
mixture, and we can also use it to measure it’s expression level.

• What it tells you:

1. The size of a protein
2. The amount of protein present (semi-quantitative)
3. In what tissue a protein is expressed

• What it doesn’t tell you:

1. Whether the protein normally exists as part of a larger complex
2. Subcellular localization of the protein
Western blot reaction
A Western blot with an epitope tagged protein
 Western blot

• Western blots of serum samples from two HIV-infected

individuals and one control subject
 Analysis of Proteins by SDS-
PAGE
• SDS (Sodium dodecyl sulphate) is a
detergent used to denature proteins and
give them a negative charge

• PAGE: Polyacrylamide Gel

Electrophoresis
 Denaturation of Proteins

Reduction of Proteins

• DTT (dithiothreitol)
• β-mercapto-ethanol
 Cellular proteins reacting with an antibody can be
characterized by immunoprecipitation of labeled cell lysates

• Cells are lysed with detergent and individual labeled cell-associated

proteins can be precipitated with MAb attached to beads.
• After unbound proteins have been washed away, the bound protein is
eluted in the detergent SDS which dissociates it from the Ab and also,
coats the protein with a strong negative charge.
• This allows the proteins migrate according to its size in PAGE
SDS-PAGE apparatus
Basics of Immunocyto (histo)
chemistry
 Definitions

• Immunohistochemistry is a technique for

identifying tissue constituents (antigens) by
means of antigen-antibody interactions.

• The site of antibody binding being identified

either by direct labeling of the antibody, or
by use of secondary antibody labeling
method.
 CELLULAR ANTIGENS

Sensory Metabolic Adhesion

 Antigen Recognition
1’Ab : primary antibody
2’Ab : secondary antibody
B : biotin
SA : streptavidin/Avidin
HRP : horseradish peroxidase or
Alkaline phosphatase
Target is antigen (protein targets)
Blocking reagent : BSA (bovine
The sandwich of antigen recognition
on tissue samples. serum albumin)
Immunohistochemistry
Immunohistochemistry uses
uses antibodies
antibodies toto detect
detect
and
and visualises
visualises antigens
antigens in
in cells
cells and
and tissues
tissues

Antibodies
Antibodiescan
canbeberaised
raisedagainst
againstalmost
almostany
anytype
typeof
of
antigen
antigen(protein,
(protein, carbohydrate,
carbohydrate, lipid...)
lipid...)

Antibodies
Antibodiesbind
bindto
toantigen
antigen in
inaaspecific
specificmanner
manner
Bound
Boundantibodies
antibodiescan
canbebedetected
detectedin
inseveral
severalways
ways

AAvery
veryimportant
importantdiagnostic
diagnosticand
andresearch
research tool
tool
ABC
ABC(Avidin
(AvidinBiotin
BiotinComplex)
Complex)--Method
Method
• Peroxidase
(intense, discrete
labeling with little or
no background. )
• Alkaline
Phosphatase
– generally not as
dense as that
achieved with a
peroxidase reaction
– Useful in
precipitating,
fluorescent
reaction product
• Glucose
oxidase
– preferred detection
system in
situations where
endogenous
peroxidase or
alkaline
phosphatase
activity presents a
problem (Tonsil).
Substrate-chromogen
solution

Enzyme-conjugated
Avidin-Biotin Complex

Biotinylated
Secondary Antibody
(anti species X)
Primary Antibody (species X)

Antigen expressing Cell

 Direct
Direct Immunofluorescence
Immunofluorescence Method
Method

Easy
Easy application,
application, only
onlyfew
fewsteps
steps

Not
Notvery
verysensitive
sensitive

Not
Notvery
veryversatile,
versatile,as
asprimary
primaryantibodies
antibodiesneed
needto
tobe
be
directly
directlylabelled
labelledwith
withfluorophore
fluorophore
Fluorochrome-conjugated
Primary Antibody

Antigen expressing Cell

 Indirect
Indirect Immunofluorescence
Immunofluorescence Method
Method
More
More steps
steps involved
involved

More
More sensitive
sensitive

More
More versatile,
versatile, as
as only
only secondary
secondary antibodies
antibodies need
need to
to
be
be labelled
labelled and
and different
different combinations
combinations of of
fluorophores
fluorophores are
are possible
possiblein
in multiple
multiple labelling
labelling
experiments
experiments
 Fluorochrome-conjugated
Secondary Antibody (anti species X)

Primary Antibody (from species X)

Antigen expressing Cell

 Indirect
Indirect Immunfluorescence
Immunfluorescence Using
Using
Biotinylated
Biotinylated secondary
secondary Antibody
Antibody

Biotin
Biotin (Vitamin
(Vitamin B)
B) binds
binds with
with high
high affinity
affinity to
to
Avidin
Avidin –– thus
thus good
good linker
linker system
system

Very
Very high
high sensitivity
sensitivity

Endogenous
Endogenous biotin
biotin may
may be
be present
present in
in tissue
tissue ––
risk
risk of
of background
background
Fluorochrome-conjugated
Streptavidin

Biotinylated
Secondary Antibody (anti species X)

Primary Antibody (from species X)

Antigen expressing Cell

 Immunofluorescence

Fluorochromes
-Fluorescein (490→517nm)
-Rhodamine (515→546nm)
-Phycoerythrin
: absorb light of one wavelength & emit
fluorescence at a longer wavelength than
fluorescein.

mIgM-producing B cells indirectly stained with rhodamine-conjugated

secondary Ab under a fluorescence microscope.
Figure A-16
• Fluorophores or fluorochromes
• Ability to absorb energy and emit light
• Two most commonly used:
– Fluorescein – green
– Tetramethylrhodamine – red

• Antibodies and bacteria are fixed on a glass-

plate.
• The surplus i.e. non-bounded antibodies are
washed out, antibody-bacteria-complexes
("sandwiches") remain.
• The "sandwich" becomes visible by adding
fluorescent anti bovine immunoglobulin which
can be seen as green light in the fluorescence
microscope.
Figure A-17

Antibodies to protein glutamic acid

decarboxylase coupled to a green dye
are shown to stain the  cells of
pancreatic islets of Langerhans. The 
cells do not make this enzyme and are
labeled with the antibodies to the
hormone glucagon coupled with an
orange fluorescent dye.
 Common Problems

Non-specific binding of primary or secondary

antibodies to tissue sample; e.g. through ionic
or hydrophobic interactions or binding of
antibodies to free amino groups:
Background staining

Cross-reactivity of antibodies with unrelated

antigens present in tissue sample
 Antigen-Antibody based
experiments: Quantitative Assays
 Radioimmunoassay (RIA)
• Competitive binding
• Uses Iodine 125 (I 125) as label
• Radioactive label competes with patient
for sites
• High radioactivity: small amount of patient
substance
• Low radioactivity: high amount of patient
substance
Radioimmunoassay
 Radioimmunoassay Competitive
• A plasma (or urine) sample that contains ADH is mixed with
the antibodies.
• A known amount of "standard" ADH that has been tagged
with radioactivity is added to the antibody mixture.
• The "standard" ADH is something that has already been
measured and that can be used to compare the unknown
quantity of natural ADH.
• A radioactive tag is added to the standard ADH so that it can
be distinguished from the natural ADH that is in the plasma.
• As the antibodies, the standard radioactive ADH, and the
plasma are mixed, one main requirement must be met:
there must be too little antibody to bind completely to
both the radioactive hormone and the natural hormone
in the fluid to be assayed.
• Therefore, the natural hormone in the plasma and the
radioactive standard hormone compete for the binding sites
on the antibody
 Enzyme Immunoassay
•ELISA
Binding of hormone to Ab reported by color rather than radiation
•Ab can be replaced by natural binding proteins such as recombinant
receptors

• Enzymes used
• Two classifications:
– Heterogeneous – separation of reactants must be
done
– Homogeneous – no separation step
• Two types:
– Competitive – known and unknown compete
– Noncompetitive – only unknown reacts
 ELISA
• 1.     You need reagent antibodies or reagent binding proteins that have
been “tagged” with an enzyme label. This means that the enzyme has
been covalently coupled to the protein reagent.
– a.     Typical enzymes include: Horseradish peroxidase or Alkaline
phosphatase
– b.     Reagent proteins include antibodies such as goat-anti-human-IgG. Or,
bacterial proteins that bind to antibodies such as Staphylococcal protein A or
Streptococcal protein G. Biotin and Avidin can also be used ---- avidin has
several high-affinity binding sites for biotin thus it can be used to bridge
molecules that have been “tagged” with biotin.

• 2.     You need some kind of “solid phase” to which proteins can stick.
This would usually be some kind of plastic microtiter plate. Thus,
antigen can be used to “naturally” coat the wells of a microtiter plate.
After you do this you would have to “block” the plate with some kind of
irrelevant protein (or detergent).

• 3. One then reacts the antigen-coated plate with appropriate enzyme-

tagged antibody. Afterwards the appropriate substrate is added and a
color-change will indicate a “positive” test.
 to detect Ab (HIV, HCV) ELISA

to detect Ag

to detect Ag

Variations in the enzyme-linked immunosorbent assay (ELISA) technique, similar to

RIA except using an Enzyme (alkaline phosphatase, horseradish peroxidase, & β-
galactosidase) and safer & less costly.
 Competitive ELISA
• Unknown antigen competes with labeled known
antigen
• Enzyme produces color reaction
 Noncompetitive ELISA
• Referred to as “Indirect” ELISA
• Antigen bound to solid phase
• Add patient, antibody will bind if present
• Add known labeled antibody
• Measure enzyme label
• Disadvantage more manipulation of test
 Immunoenzymometric Assay
• Noncompetitive ELISA
• Detects unknown antigen by means of
excess labeled antibody
 Sandwich or Capture Assays
• Used for antigens with multiple epitopes,
ie, HCG
• Antibody to one epitope fluid, antibody to
second epitope fixed.
• Enzyme label used to detect reaction
 ELISA

The ELISPOT assay, a

modification of the ELISA assay to
determine quantitatively
the # of cells in a population that are
producing specific Ab or cytokine.

-> precipitates & forms a spot only on the areas of the

well where cytokine-secreting cells had been
deposited.
 Advantages
• Non-radioactive
• High specificity
• Color change or florescence allows to
use photometroscopy
• Amplification of minute levels allows
for high sensitivity
• Qualitative and Quantitative
• Used to find blood serum antigens
۞Separation techniques have the advantage that they give a
high yield more quickly than cloning, although not with the
same purity.

۞ The more successful separation techniques depend on

differences in
(1) cell density (specific gravity),
(2) affinity of antibodies to cell surface epitopes,
(3) cell size, and
(4) light scatter or fluorescent emission as sorted by flow
cytometry.

۞The first two techniques involve low level technology and are
inexpensive, whereas the next two techniques involve high
technology and are expensive.
 2. Antibody-based techniques

۞There are a number of techniques that rely on

the specific binding of an antibody to the cell
surface.

۞The techniques all depend on the specificity of

the selected antibody and the presentation of
the correct epitope on the cell surface of living
cells as confirmed by immunostaining and/or
flow cytometry.
Magnetic sorting
۞ Magnetic sorting uses a
specific antibody raised
against a cell surface
epitope conjugated to
ferritin beads (Dynabeads).

۞ When the cell suspension

is mixed with the beads
and then placed in a
magnetic field, the cells
that have attached to
Dynabeads are drawn to
the side of the separating
chamber.

۞ The cells and beads are

releases when the current
is switched off, and the
cells may be separated
from the beads by
Trypsinization or vigorous
pipetting.
Figure A-26
 Magnetic Cell Sorting

۞ Several cell types have been separated by this method including stem cell
purification by negative sorting, purging bone marrow of leukemic cells, and
isolation of kidney tubular epithelium.

۞ The use of immunomagnetic microbeads allows the cells to be cultured or

processed through further sorting procedures without the need to remove the
beads as in Miltenyi. The method is therefore particularly useful for a positive
sorting.
What is Flow Cytometry?
• Cytometry refers to the measurement of physical/chemical characteristics of
cells or other biological particles.

• Flow Cytometry is the process whereby such measurements are made upon
cells/particles as they pass through a measuring apparatus (hopefully in single
file) suspended in a fluid stream.

• Flow Sorting (Flow Cytometric Cell Sorting) extends flow cytometry with
the additional capacity to divert and collect cells exhibiting an identifiable set of
characteristics either mechanically or by electrical means (Flow Cytometric
Analysis).

• FACS - Fluorescence Activated Cell Sorting. FACS is a trademark of

Becton Dickinson Immunocytometry Systems (BDIS). All FACS instruments
are BDIS systems, but not all cytometers are FACS.
 3. Fluorescence-activated cell sorting

Flow
Cell

Sheath and
Waste Tanks Sample
Inlet
“FACS” adds the ability to detect fluorescent probes attached to cells
and sort them if desired.
 Measurements in Flow Cytometry

Laser

Forward Active Light

Scatter Sensor

Side Scatter Light (90°) Sensor

 Light Scatter Gating
Side scatter SSC = Side Scatter Projection
granularity

1000
Neutrophils

Forward Scatter Projection

Scale
800
1000
Forward Scatter

200
100
600

50
40
30 Monocytes
400

20
15
8
Lymphocytes
200

Forward scatter
0

0 200 400 600 800 1000

90 Degree Scatter FSC =

cell size
Figure A-25 part 2 of 2
Key Transition Points in the Cell Cycle
 Channel number (Fluorescence)

Printout from FACS: Murine erythroleukemia (Friend cells) cells were fixed in methanol
as a single suspension and stained with chromomycin A3. The suspension was
then mixed with chicken RBC, fixed, and stained in the same way and run through
FACS. As fluorescence is directly proportional to the amount of DNA per cell, the
trace gives a distribution analysis of the cell population by DNA content. The lowest
DNA content is found in chicken RBCs included as a standard. The major peak
around channel 100 represent those cells as the G1 phase of the cell cycle. Cells
around channel 200 therefore represent G2 and Metaphase cells (with double the
amount of DNA per cell), and cells in intermediate channels are in the S (DNA
synthetic) phase. Cells accumulated in channel 250 are those for which the DNA
value is off scale or those that have formed aggregates.
 Uses for Multiple Fluorescence Parameters

• Phenotyping (Cell Surface Antigens)

– 168 CD Cell Surface Antigens
– Many functional populations require 5 or more surface
markers to be fully distinguished
• Functional Assays
– Cell Cycle (PI, BrdU, Intracellular Cyclins)
– Apoptosis (AnnexinV, Active Caspase-3)
– Ca++ Flux [Indo-1, Fura]
• Cytokine Production
• Intracellular Signaling (Rb phosphorylation)
• Gene Reporter [Molecular] Assays
– GFP, BFP, YFP, NFP Expression
– LacZ Expression
Advantages/ Disadvantages of Using More Colors

• Advantages
– Save Time, Reagents, Samples-
• (1) 6-color stain = (15) 2-color stains
– Exponential increase in information
• Data from (1) 6-color stain » (15) 2-color stains
– identify new/rare population (<0.05%)
– internal controls
• Problems
– Must carefully choose combinations of
fluorochrome conjugates
• All reagents not available in all colors
– Greater potential for errors in compensation
• Proper controls required
Cell culture and experimental models
The complexity of the cellular interactions that
generate an immune response has led immunologists
to rely heavily on various types of in vitro cell-culture
Systems

A variety of cells can be cultured, those include

- primary lymphoid cells,
- cloned lymphoid cell lines and
- hybrid cells.

Primary lymphoid cell cultures can be obtained by

Isolating lymphocytes directly from blood or lymph or
from various lymphoid organs by tissue dispersion.

Cloned Lymphoid Cell Lines are normal lymphoid

Cells, except that have undergone transformation
induced by chemical carcinogens or viruses
can be propagated indefinitely in tissue culture; thus,
they are said to be immortal. Such cells are referred
to as cell lines.

hybrid cells immunologists fuse normal B

or T lymphocytes with tumor cells, obtaining
hybrid cells
 Experimental Animal Models
• study of the immune system in vertebrates requires suitable
animal models
• choice of an animal depends on its suitability for attaining a
particular research goal
• For large amount of antiserum we may use the following animals
rabbit, goat, sheep, or horse
• For the need of vaccine production and signaling research
purpose we use animals which includes mice, rabbits, monkeys,
chimpanzees, or baboons.
• For most basic research in immunology, mice have been the
experimental animal of choice for the following reasons
- easy to handle,
- genetically well characterized
- have a rapid breeding cycle.
- The immune system of the mouse has been characterized
more extensively than that of any other species.
• Inbred strains: Selective inbreeding of littermates for
more than 20 generations leads to production of inbred
strains.
– Genetically identical, syngeneic
– MHC class I and II were identified using these strains
• Adoptive Transfer of T cells is usually performed using
genetically identical donor and recipients (inbred strains).
• Severe Combined Immunodeficiency Disease (SCID) is a
disorder in which B and T cells fail to develop
– In 1980s, an inbred strain of mice spontaneously developed an
autosomal recessive mutation resulting in SCID.
– SCID-chimeras (engrafted with human hematopoietic stem cells).
A valuable animal model for studying the development of
lymphoid cells.
An autosomal recessive mutation resulting
in severe combined immunodeficiency
disease (SCID) developed
Spontaneously in a strain of mice
called CB-17.

These CB-17 SCID mice fail to develop

mature T and B cells and consequently
are severely compromised immunologically.
This defect is due to a failure in V(D)J
recombination. SCID mice must be
housed in a sterile (germ-free)
environment, because they
cannot fight off microorganisms of even
low pathogenicity.

The absence of functional T and B cells

enables these mice to accept foreign cells
and grafts from other strains of mice or
even from other species.
• Thymectomized and Congenically Athymic (Nude) mice
– Mice are neonatally thymectomized, irradiated and then
reconstituted with syngeneic bone marrow. These mice fail to
develop T cells
– Mice homozygous for a mutation in a gene called nu also fail to
develop mature T cells resulting in athymic phenotype.
– The above two models are useful in studying T cell development.
– Useful in propagation of tumor cell lines and freshly harvested
tumor tissue from other strains because these mice lack T cells

• Transgenic mice
– Injecting a cloned gene (transgene) into fertilized mouse eggs.
– It can be transmitted to offspring because it is in both somatic and
germline cells.
– By constructing a transgene with a particular promoter, it is
possible to control the regulation of transgene
• Knock-out Mice
– Sometimes, it is of interest
to determine how the
removal of a particular gene
product affects the immune
system
Procedure for
– Gene-targeting method producing
allows replacement of a transgenic
normal gene with a mice
mutated/disrupted gene to
get knock-out mice
Analysis of gene expression
The transcriptional activity of genes is regulated by promoter and enhancer
sequences.

The activity of enhancer and promoter sequences is controlled by

transcription factors, which are DNA-binding proteins.

These proteins bind to specific nucleotide sequences within promoters and

enhancers and act either to enhance or suppress their activity.

Enhancer and promoter sequences and their respective DNA-binding

proteins have been identified by a variety of techniques, including DNA
footprinting, gel-shift analysis, and the CAT assay.
DNA Footprinting Identifies the Sites
Where Proteins Bind DNA

-cloned DNA fragment containing a putative

enhancer or promoter sequence is first
radiolabeled at the 5 end with 32P.

-one fraction is incubated with a nuclear

extract containing a DNA-binding protein

-Other fraction DNA fraction is not incubated

with the extract.

-Both DNA samples are then digested with a

nuclease or a chemical that makes random
cuts in the phosphodiester bonds of the
DNA, and the strands are separated.

- In the absence of DNA-binding proteins,

a complete ladder of bands is obtained on
the electrophoretic gel

-When a protein that binds to a site on the

DNA fragment is present, it covers some of the
nucleotides, protecting that stretch of the DNA
from digestion
Gel-Shift Analysis Identifies DNA-Protein
Complexes

-When a protein binds to a DNA fragment,

forming a DNAprotein complex, the electrophoretic
mobility of the DNA fragment in a gel is reduced,
producing a shift in the position of the band
containing that fragment.

-In this technique, radioactively labeled cloned

DNA containing an enhancer or a promoter
Sequence is incubated with a nuclear extract
containing a DNAbinding protein.

-The DNA-protein complex is then

electrophoresed and its electrophoretic mobility
is compared with that of the cloned DNA alone.

-A shift in the mobility indicates that a protein is

bound to the DNA, retarding its migration on the
electrophoretic gel.
CAT Assays Measure Transcriptional Activity

-DNA construct consisting of the promoter of

interest and the reporter gene encoding
Chloramphenicol acetyltransferase (CAT) is
introduced (transfected) into eukaryotic cells.

-If the promoter is active, the CAT gene will be

transcribed and the CAT enzyme will be produced
within the transfected cell.

-The presence of the enzyme can easily be

detected by lysing the cell and incubating the cell
lysate with [14C] chloramphenicol and acetyl-CoA.

-If present, the CAT enzyme will transfer the acetyl

group from acetyl-CoA to the chloramphenicol,
forming acetylated chloramphenicol, which can be
easily detected by thin-layer chromatography.
Microarray