Immunology – II

BC402
Immunological Methods and
Applications

ASHUTOSH SINGH
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Suggested Reading:

Immunology by Barbara Anne Osborne and Janis Kuby

Terminologies
 In vivo
 Involve whole animal

 In vitro
 Defined populations of immune cells are studied under controlled lab
conditions

 Study of immune system requires suitable animal models

 For vaccine development – is the animal model susceptible to the disease?
 Mouse most often used
 Inbred strains reduce variation caused by differences in genetic
backgrounds
o 20 or more generations of brother-sister mating
 Have to abide by IACUC guidelines

 Adoptive Transfer
 Immune system of model animal can be eliminated
 Replaced with immune cells of animal to be studied
Polyclonal Antibodies Are Secreted by Multiple Clones of
Antigen-Specific B Cells

 Use of polyclonal antibodies

 Immunizing animal (mouse, rabbit) or human with antigen one or
more times
 Taking blood samples, purifying the antibodies from the serum
 Results in a mixture of antibodies directed towards variety of different
epitopes
 Disadvantages:
o Ill-defined cross-reactivities with related antigens
o Range of cross-reactivity to desired antigen might vary from
bleed to bleed
o Animal might die, causing you to start over
A Monoclonal Antibody Is the Product of a Single Stimulated
B Cell

 Use of monoclonal antibodies

 Product of single, stimulated B cell
o Supply of antibody specific for one epitope
 Uses:
o Can be specific for specific targe cells and conjugated to toxins
o More sensitive/specific ELISAs
 Cell Culture Systems
 Cells are cultured and studied
 Specialized media
 Can be used for:
o Testing effects of contaminants on immune cells
o Testing drugs
o Producing monoclonal antibodies

 Cell line
 Cells that have been transformed – propagate indefinitely
(cancerous cells)
 •Köhler and Milstein:
used myeloma cells
lacking the enzyme
hypoxanthine guanine
phosphoribosyl
transferase (HGPRT).
•The mutant tumor cells
and tumor-tumor hybrids
would be unable to
synthesize new DNA by
either the salvage or the
de novo pathways and
would eventually die.
•Hybridomas formed by
fusion between B cells
and tumor cells, the B-
cell parent would provide
the HGPRT, and so these
hybrids would survive in
the selection medium.

The conventional polyclonal antiserum produced in response to a complex antigen contains a mixture of
monoclonal antibodies, each specifi c for one of the four epitopes shown on the antigen (inset). In contrast, a
monoclonal antibody, which is derived from a single plasma cell, is specific for one epitope on a complex antigen.
The outline of the basic method for obtaining a monoclonal antibody is illustrated here.
 Fusion producing
hybridoma

A hybridoma possesses the

immortal growth properties of the
myelomacell parent and secreted
the unique antibody produced by
the B-cell parent.
ABZYMES

mAbs can specifically bind and stabilize the transition state

of a chemical reaction, thus directly mimicking the activity
of enzymes.
Such antibodies with enzyme-like activities are referred to as
abzymes.
 Immunoprecipitation- Based Techniques
Immunoprecipitation Can Be Performed in Solution

When bi or multivalent antibodies are mixed in solution with antigen, the antibodies can form cross-linkages
with two or more antigen molecules, leading to the formation of a cross-linked precipitate (middle panel of graph).
Precipitate formation requires that neither antigen (left panel in graph) nor antibody (right panel in graph)
molecules are in excess.
In either of these two cases, primarily monovalent binding takes place.
Immunoprecipitation of Soluble Antigens Can Be Performed in
Gel Matrices
Ouchterlony double immunodiffusion
Whether 2 Ag share common epitopes?
 Agglutination Reactions

Hemagglutination Reactions Can Be Used to Detect Any Antigen Conjugated

to the Surface of Red Blood Cells

Hemagglutination Inhibition Reactions Are Used to Detect the Presence of Viruses

and of Antiviral Antibodies

Bacterial Agglutination Can Be Used to Detect Antibodies to Bacteria

Radioimmunoassays (RIA)
Are Used to Measure the Concentrations of Biologically Relevant Proteins (Antigens) and
Hormones in Body Fluids
Gamma Counter
 From these data, a standard
binding curve, like the one
shown in red, can be
drawn.
 The samples to be assayed
(the unknowns) are run in
parallel.
 After determining the ratio
of bound to free antigen in
each unknown, the antigen
concentrations can be read
directly from the standard
curve.
 The main drawbacks to radioimmunoassay are the
expense and hazards if preparing and handling the
radioactive antigen.
 Both 125I or 131I emit gamma radiation that requires
special counting equipment;
 The body concentrates iodine atoms — radioactive or
not — in the thyroid gland where they are incorporated
in thyroxine (T4).
 Despite these drawbacks, RIA has become a major tool in
the clinical laboratory where it is used to assay
 plasma levels of:
 most of our hormones;
 digitoxin or digoxin in patients receiving these drugs;
 certain abused drugs
 for the presence of hepatitis B surface antigen (HBsAg) in
donated blood;
 anti-DNA antibodies in systemic lupus erythematosus (SLE).
ELISA Assays Use Antibodies or Antigens Covalently Bound to Enzymes
 Protein Biochemistry
 Biotin labels
 Biotin – small molecule that can be bound to antibody
 Used in ELISA
 Reacts with avidin to produce color change
 Schematic representation of the avidin-biotin complex (ABC) staining method.

The Labeled Streptavidin Biotin (LSAB) Staining Method

 Let’s say I’m trying to develop an ELISA to detect HS (harbor seal) IgG antibody levels in
serum
 Need a monoclonal antibody specific for HS IgG
 So, I isolate HS IgG using column chromatography, inject mouse, mouse produces anti-
IgG (remember there are idiotypic differences between IgG of mouse and another
species)
 Extract spleen (there are some B cells producing antibodies specific to the HS IgG I
innoculated with); perform fusion to create hybridomas
 After a few weeks, I have some living hybridomas – perform ELISA to see if they are
producing antibody
 Isolate the hybridomas (want to make sure I only have clones from 1 B cell)
 My ELISA tells me they are producing anti-HS IgG but I want to see if the epitope is on
the light or heavy chain
o Coat plate with isolated HS IgG, then add media from monoclonals containing
anti-HS IgG, followed by biotinylated anti-mouse
 Therefore, I can use a Western blot to see this
o Next 2 slides
 Protein Biochemistry
 Gel Electrophoresis
 SDS-PAGE
 SDS is a detergent, binds to proteins and destroys tertiary and
secondary structure
 Proteins can be separated according to molecular weight
o Separation of antibody classes (different heavy chains, separation of
light and heavy chains)
o Run IgG I’m interested in looking at (HS-IgG I injected into
mouse)
o This can then be used in Western Blot (next slide)
Western blotting uses
antibodies to identify
protein bands following
gel electrophoresis.
ELISPOT Assays Measure Molecules
Secreted by Individual Cells
 Protein Biochemistry
 X-ray crystallography
 Limit of light microscopy is resolution
 X-rays are transmitted through crystallized
protein
 Different atoms will scatter the x-rays
differently
 Pattern contains information of position of
atoms within the molecule
 Detector records pattern of spots
 Mathematical deduction leads to calculation
of structure
Surface Plasmon Resonance Is
Now Commonly Used for
Measurements of Antibody
Affinity
 Complement Fixation Test
 In the positive test : The available
complement is fixed by Ag-Ab complex and no
hemolysis of sheep RBCs occurs. So the test is
positive for presence of antibodies.

 In the negative test : No Ag-Ab reaction

occurs and the complement is free. This free
complement binds to the complex of sheep
RBC and it’s antibody to cause hemolysis,
causing the development of pink color.

 Controls should be used along with the

test to ensure that
 Antigen and serum are not anti
complimentary
 The appropriate amount of complement is
used and
 The sheep red blood cells do not undergo
autolysis
MICROSCOPY: Staining
MICROSCOPY: Immunofluorescence-Based Imaging Techniques
MICROSCOPY: Confocal Microscopy
Flow Cytometry: Fluorescence Activated Cell Sorter (FACS)
Cell Complexity

Cell Size
 Fluorescent Technology
 Green fluorescent protein
 Isolated from bioluminescent jellyfish, naturally occurring
 Can be used to visualize live cells
Cell Cycle Analysis
Tritiated (3H) Thymidine Uptake
Was One of the First Methods Used to Assess Cell Division.
3H thymidine uptake assays were the first to be used routinely to measure cell
division in lymphocyte cultures.
They rely on the fact that dividing cells synthesize DNA at a rapid pace, and
radioactive thymidine in the culture fluid will therefore be quickly incorporated into
high molecular weight DNA.

Colorimetric Assays
The tetrazolium compound MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5
diphenyltetrazolium bromide, a yellow tetrazole) is reduced by metabolically active
cells to form insoluble, purple formazan dye crystals (Abs at 570nm).
Bromodeoxyuridine-Based Assays
Use Antibodies to Detect Newly Synthesized DNA.

When introduced into cells,

bromodeoxyuridine (BrdU) is rapidly
phosphorylated to bromodeoxyuridyl
triphosphate (an analogue for
deoxythymidine triphosphate) and is
incorporated in its place into newly
synthesized DNA.

Cells that divide following BrdU

incorporation can then be identified using
antibodies to BrdU.
Propidium Iodide Enables Analysis of the Cell Cycle Status of Cell Populations

G1 cells will have half the DNA of G2 cells, or cells about to undergo mitosis, and cells
that are currently replicating DNA and are therefore in S phase will have an intermediate
value. Apoptotic cells and fragments that have begun to break down their DNA will
appear as events with less than G1 amounts of DNA.
Carboxyfluorescein Succinimidyl Ester Can Be Used to Follow Cell Division
The diacetyl groups enable the CFDASE to enter the cell, and are then cleaved by
intracellular esterases.
CFSE remains trapped within the cytoplasm.
In the cytoplasm, molecules of CFSE are efficiently and covalently attached to
intracytoplasmic proteins, with the succinimidyl ester acting as a leaving group.
The amount of fluorescence emitted is cut in half each time the cell divides.
Cell Death Assays
The 51Cr Release Assay Was the First Assay Used to Measure Cell Death
51Cr release assay: for measuring cytotoxic T cell- and natural killer cell-mediated
killing.
Target cells incubated with sodium 51chromate and then the killer cell.
Modern Alternative: CFSE (carboxyfl uorescein succinimidyl ester) release upon
killing.

Fluorescently Labeled Annexin V Measures Phosphatidyl Serine in the

Outer Lipid Envelope of Apoptotic Cells

Phosphatidyl serine flips from the interior to the exterior side of the plasma
membrane phospholipid bilayer.
Annexin V is a protein that binds to phosphatidyl serine in calcium dependent manner

Caspase Assays Measure the Activity of Enzymes Involved in Apoptosis

Caspase 8, Assay kits available (fluorescence or western blot based).
The TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) Assay
Measures Apoptotically Generated DNA Fragmentation
Assay is based on terminal deoxynucleotidyl transferase (TdT) to add bases onto the broken
ends of DNA sequences in a non templated manner.
The classic variation of the TUNEL method uses TdT to add BrdU to fixed and permeabilized
cells.
BrdU is incorporated into the newly synthesized DNA, and is then detected with fluorescently
labeled anti-BrdU antibodies
Biochemical Approaches Used to Elucidate Signal Transduction
Pathways
Recombinant DNA
Technology
 Restriction enzymes cleave
DNA at precise sequences
 DNA sequences are cloned into
vectors
○ Virus
 If it’s a bacteriophage, it can
then infect bacteria and the
bacteria will express
inserted gene
○ Plasmid
 Gene of interest is inserted
into plasmid containing
antibiotic resistance gene,
incubated with bacterial
cells, if bacteria uptake
plasmid they will be able to
grow on medium with
antibiotic
Recombinant DNA Technology
 Cloning of cDNA and genomic DNA
 Messenger RNA isolated from cells can be transcribed into
complementary DNA
 This can be inserted into vector and then expressed
 cDNA library
 Expressed genes of cell
 Recombinant DNA Technology
 Southern Blotting
 Gene Transfer
 Common technique
 Retrovirus – replace viral structural gene with clone gene to be
transfected
 Virus is now used as vector to insert new gene into cultured cells

 Inserting these transgenes into mouse embryos allows researchers to

study effects of immune system genes in vivo

 Gene Transfer
 Knockout mice
 Replace normal gene with mutant allele
Animal models
 Microarrays
 Assess differences in gene expression between cell types
 Can scan large #’s of mRNAs
 Procedure
 mRNA isolated, cDNA synthesis is initiated
 First strand of cDNA is labeled with tag
 Labeled cDNA is then hybridized with nucleic acid affixed in microarray