Immunological Techniques I

Immunology and Immunotherapy
 Immunodiffusion
 Immunoelectrophoresis
 ELISA
 RIA
 FACS
 Hybridoma
IMMUNODIFFUSION
 Diffusion of antigen or antibody through a semisolid
medium, (agar or agarose gel), with the result being a
precipitin reaction- precipitation of an antibody out of
solution upon antigen binding
 Varying amounts of soluble antigen are added to a
fixed amount of serum containing antibody.
 First quantitative assay for antibody
• Ouchterlony double immunodiffusion (Agar gel immuno
diffusion/ Passive double immunodiffusion)- Gold standard for
detection of extractable nuclear antigens
• Radial immunodiffusion (Mancini immunodiffusion/ Single radial
immunodiffusion assay)- Used to determine quantity of an antigen
PRECIPITIN REACTION
Zone of Antibody Excess
• Each antigen molecule bound extensively by antibody; crosslinked
to other antigen molecules
• Average size of antibody-antigen complex is small
• Rare cross-linking between antigen molecules by antibody
Zone of Equivalence
• Optimal formation of precipitin complexes
• Extensive lattices of antigen and antibody formed by cross-linking
Zone of Antigen Excess

• Average size of antibody-antigen complex is small
• Rare cross-linking between antigen molecules by antibody
OUCHTERLONY
 Developed by Örjan Thomas
Gunnarson Ouchterlony, a Swedish
bacteriologist, in 1948
 Can simultaneously monitor multiple
antibody-antigen systems
Disadvantages
 Time  Interpretative expertise required
 Need for reagent sensitivity  Selectivity validation
RADIAL IMMUNODIFFUSION
 Developed by Giuliana Mancini in 1948
 Detection and quantitation of all classes of immunoglobulins,
complement, ceruplasmin, transferrin, and other serum components
 Antibody mixed with agar, poured onto a glass slide and allowed
to harden
 Known concentrations of antigen placed into a series of wells
from which they diffuse radially into the antibody containing agar
 Visible circles of precipitate form around each well - diameter
proportional to antigen concentration
 Concentration in unknown sample determined from a standard
curve constructed from the ring diameters formed by the known samples
ELISA and RIA
 Method for quantifying antigen concentration with
sensitivity and specificity using an indicator molecule
 Used for detection and quantification of peptides, proteins,
antibodies and hormones
 Radioactive indicator molecule- Radioimmunoassay (RIA)
 Indicator molecule covalently linked to an enzyme -
Enzyme-linked immunosorbent assay (ELISA)
DIRECT DETECTION
Advantages:
 Quick - Only one antibody; fewer steps
 Cross-reactivity of secondary antibody eliminated
Disadvantages:
 Antibody immunoreactivity might be affected by labeling
 Time-consuming and expensive
 No flexibity in choice of primary antibody
 Minimal signal amplification
 Rarely used for RIA/ELISA; used commonly for immuno-

histochemistry of cells/tissues
INDIRECT DETECTION
Advantages:
 Availability of wide variety of labeled secondary antibodies
 Versatile; same secondary antibody may be used for many
primary antibodies raised in the same species
 Maximum immunoreactivity of primary antibody
 Increased sensitivity due to signal amplification
 Use of different visualization methods for same primary
antibody
Disadvantages:
Possibility of cross-reactivity; non-specific signal
Extra incubation step
SANDWICH/CAPTURE ELISA/RIA
Antigen “sandwiched” between the “capture antibody”

and the “detection antibody”
The capture antibody and detection antibody recognize
different epitopes on the same antigen
The detection antibody is usually tagged with the
detection conjugate to avoid non-specificity
Advantages: More specific, highly sensitive
Disadvantages: Very expensive, more time consuming
COMPETITIVE ELISA/RIA
 Unlabeled antibody is incubated in the presence of its antigen

 Bound antibody/antigen complexes added to an antigen coated well
 More antigen in the sample, the less antibody will be able to bind to
the antigen in the well, hence "competition."
 Detection using labeled secondary antibody
 Higher sample antigen concentration, weaker signal
 Ability to use crude or impure samples

 Sometime, enzyme-linked antigen rather than enzyme-linked
antibody is used; more antigen in the sample, the less labeled
antigen is retained in the well and the weaker the signal
FACTORS AFFECTING ELISA/RIA
o Plate Selection
o Plate Coating
o Blocking and Wash Buffers
o Antibodies
o Detection Strategies
IMMUNOELECTROPHORESIS
Separation and characterization of proteins based on
electrophoresis and reaction with antibodies
Electrophoresis: Sorting of molecules based on size and charge

• Immunoblotting/Western Blotting
• Crossed immunoelectrophoresis
• Rocket immunoelectrophoresis
• Affinity Electrophoresis
WESTERN BLOTTING
• Originated from the laboratory of George Stark at Stanford
• Name given by W. Neal Burnette (1981), play on the name Southern
blot a technique for DNA detection developed earlier by Edwin
Southern (Northern blotting-detection of RNA; Eastern blotting-
detection of post-translational modification of proteins
Steps in Western Blotting
• Sample Preparation
• Electrophoretic separation of proteins
- One dimensional - denaturing (SDS-PAGE) or native
- Two-dimensional
• Transfer to membrane(Polyvinylidene fluoride or nitrocellulose)
• Blocking • Probing - Use of Primary and Secondary Abs
• Visualization
SAMPLE PREPARATION & ELECTROPHORESIS
DETECTION OF IMMUNOCOMPLEXES
DETECTION OF IMMUNOCOMPLEXES
 Colorimetric detection
 Chemiluminescent detection
 Radioactive detection
 Fluorescent detection
SECONDARY PROBING
CROSSED IMMUNOELECTROPHORESIS
 Also called two-dimensional quantitative immunoelectrophoresis
 Proteins separated during the first dimension electrophoresis;
electrophoresed into an antibody-containing gel in the second dimension
 Immunoprecipitation takes place during the second dimension
electrophoresis; immunoprecipitates have a characteristic bell-shape
 Each precipitate represents one antigen, the position being dependent
on the amount of protein as well as the amount of specific antibody
 Higher sensitivity and resolving power
 Studies of proteins in biological fluids
and extracts
ROCKET IMMUNOELECTROPHORESIS
 Also called electroimmunoassay or electroimmunodiffusion
 Comparison of the sample of unknown concentration with a series of
dilutions of a known concentration of the protein
 Samples loaded side-by-side in small circular wells along the edge of
an agarose gel that contains the monospecific antibody
 During electrophoresis, antigen starts to interact with the antibody; the
Antigen moves along the gel till precipitin is formed as the
antigen:antibody equivalence is attained
 Distance moved through the gel is directly
proportional to the amount of antigen
 Used for detection of antigens in highly complex
protein mixtures (serum sample, tissue extract,
urine sample, cerebrospinal fluid)
AFFINITY IMMUNOELECTROPHORESIS
 Based on changes in the electrophoretic pattern of proteins through

biospecific interaction or complex formation with other macromolecules
 Used for estimation of binding constants, as for instance with lectins

or for characterization of proteins with specific features like glycan
content or ligand binding
FLOW CYTOMETRY
• Cytometry: Measurement of physical/chemical characteristics of
cells or other biological particles.
• Flow Cytometry: 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.
PRINCIPLE OF FLOW CYTOMETRY
• Beam of single wavelength light (usually laser) directed onto

hydrodynamically-focused stream of fluid
• Detectors:
Forward Scatter (FSC): In line with light beam (cell volume)
Side Scatter (SSC): Perpendicular to the beam (inner complexity)
Fluorescence Detectors
COMPONENTS OF FLOW CYTOMETRY
 Flow Cell - Liquid stream which carries and aligns the cells so
that they pass through light beam in a single file
 Measuring system - Measurement of impedence/ conductivity and
optical systems - lamps (mercury) high power-water cooled lasers
(argon, krypton, dye laser) and/or low-power air-cooled lasers
 Detector and Analogue-to-Digital Conversion (ADC) System-
Generates FSC, SSC, fluorescence signals from light to electrical
 Amplification System- Linear or logarithmic
 Computer for analysis of signals
4 lasers and 18 fluorescence detectors

Flow Cell
Injector
Tip Sheath
fluid
Fluorescence
signals
Focused laser
beam
MEASUREMENTS IN FLOW CYTOMETRY
• Electronic Cell Volume

– detect and measure the volume of particles as they pass
through a small orifice
– viable cells are better insulators than fixed/dead cells
• Light Scatter
– all objects passing through a laser beam in a cytometer
will scatter light
– large objects will scatter more light in the forward
direction than small objects
– light scatter signals are commonly used to trigger data
acquisition
DATA ANALYSIS IN FLOW CYTOMETRY
o Data generated can be plotted in 1,2 or 3 dimensions

o Regions on the plots may be sequentially separated based
on fluorescence intensity by creating a series of subset
extractions, termed “gates”
o Compensation- Both electronic and computational due to
overlap of the flurescent dyes’ emission spectra
o Softwares: Flowjo, Cellquest, WinMDI
o Automated population identification: FLOCK, FLAME,
FLOWCLUST
FLUORESCENT LABELS
Blue Argon Laser (488 nm)
Green (usually labelled FL1): FITC, Alexa Fluor 488, GFP, CFSE,
CFDA-SE, DyLight 488
Orange (usually FL2): PE, PI
Red channel (usually FL3): PerCP, PerCP-Cy5.5, PE-Alexa Fluor
700, PE-Cy5 (TRI-COLOR), PE-Cy5.5
Infra-red (usually FL4); PE-Alexa Fluor 750, PE-Cy7
Red diode laser (635 nm)
Violet laser (405 nm)

MEASURABLE PARAMETERS IN
FLOW CYTOMETRY
• Cell volume, morphological compexity, viability
• DNA,(cell cycle, kinetics, proliferation, copy number)
• Cell pigments (chlorophyll, phycoerytrin)
• Chromosome analysis and sorting
• Protein expression, localization, modifications
• Cell surface, intracellular, nuclear antigens
• In vivo transgenic expression
• pH, intracellular ionized calcium, magnesium, membrane potential
• Membrane fluidity • Oxidative burst

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Immunology and Immunotherapy

Zone of Antigen Excess

 Rarely used for RIA/ELISA; used commonly for immuno-

Antigen “sandwiched” between the “capture antibody”

 Unlabeled antibody is incubated in the presence of its antigen

 Ability to use crude or impure samples

Electrophoresis: Sorting of molecules based on size and charge

 Based on changes in the electrophoretic pattern of proteins through

 Used for estimation of binding constants, as for instance with lectins

• Beam of single wavelength light (usually laser) directed onto

4 lasers and 18 fluorescence detectors

• Electronic Cell Volume

o Data generated can be plotted in 1,2 or 3 dimensions

Red diode laser (635 nm)

Violet laser (405 nm)

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