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
