Immunological Techniques

Presented by
Vaibhav Jain

Department of Biophysics
National Institute of Mental Health and Neurosciences
 Overview
Immunological techniques include both experimental methods to study
the immune system and methods to generate or use immunological
reagents as experimental tools.

Most common immunological methods relate to the production and use

of antibodies to detect specific proteins in biological samples are:

Immunoprecipitation

Immunoelectrophoresis

RIA and ELISA

Immunostaining

Immunoblotting

Flow Cytometry
Sensitivity of various Immunotechniques
Immunoprecipitation
 Immunoelectrophoresis
Immunoelectrophoresis combines
electrophoresis with immunodiffusion.

The combination of these two

techniques increases resolution and
enables to analyze interaction of
antibodies with multiple antigens.

Fig. Immunodiffusion Principle

i. Qualitative Immunoelectrophoresis

ii. Crossed Immunoelectrophoresis

iii. Rocket Immunoelectrophoresis

iv. Two - dimensional Immunoelectrophoresis

Fig. Immunoelectrophoresis arc
Radioimmunoassay (RIA)
1960 Yalow and Berson (Nobel Prize)
Very sensitive: can detect material present at
concentrations of <0.001 micrograms/ml.
Takes advantage of protein binding to plastic
of tissue culture dish (solid-phase).
Generate standard curve with known amounts
of unlabeled antigen.
Measure unknown using calibration curve.
RIA to detect hepatitis B virus
in blood sample

The antibody is immobilized on the

walls of microtiter wells and the
amount of bound antigen
determined.

can detects hepatitis virus in 1

microliter of blood.

used for screening blood for

transfusions.
 Enzyme-linked immune sorbant assay (ELISA)

ELISA assays are similar in principle

to RIAs but, instead of
radioisotopes, they use antibodies
or antigens covalently bound to
enzymes.

The conjugated enzymes are

selected on the basis of their ability
to catalyze the conversion of a
Advantages:
substrate into a colored, fluorescent, Match the sensitivity of RIAs
Safer and less costlier.
or chemiluminescent product.
Source: Kuby Immunology, 8th ed.
ELISA based Pregnancy Test
 Enzyme-Linked Immuno-Spot (ELISPOT)

ELISPOT assay allows visualization

of the secretory products of
individual activated cells.

Each spot that develops in the assay

represents a single reactive cell.

Provides both qualitative and

quantitative information.

The ELISPOT assay can be used to determine the number of cells within a
sample that are secreting a particular cytokine.
 Western Blotting
Principle:
Western blotting identifies and
provides preliminary quantitation of a
specific protein in a complex mixture of
proteins.

Western blotting uses antibodies to

Figure: Western blot using an
identify protein bands following gel antibody that recognizes proteins
modified with lipoic acid.
electrophoresis, thus combines the
resolving power of gel electrophoresis
with the specificity of antibodies.
Procedure:

Sigma-Aldrich
Gel electrophoresis separates proteins by size,
so Western blot not only provides quantitation,
but also provides information about the
molecular weight of antigen.

Source: Kuby Immunology, 8th ed

 Flow Cytometry

Flow cytometry is an analytical

technique (Leonore and
Leonard).

Quantifies the frequencies of

cells binding to fluorescent
antibodies and scattering light
in characteristic ways.

Quantitative: Sensitive: Flexible:

Accurately determine Analysis can be Fluorescent labeled
relative fluorescent performed with antibodies specific for
(protein) levels on <104 cells. many cell surface proteins
individual cells. are readily available.
Instrument Overview:
Schematics of Flow Cytometer
Visualizing Flow cytometer Data

Fig. 2D Scatter Plot to differentiate between

leukocyte subpopulations in the blood.

Thermo Fisher
 Fluorescence activated Cell sorting (FACS)

Flow cytometer is adapted to sort cell

subpopulations on the basis of fluorescence
and light scattering, referred to as a
Fluorescence Activated Cell Sorter (FACS).

For example, a cell expressing one cell

marker may be detected using an FITC-
conjugated antibody, another cell type
expressing a different marker could be
detected using a PE-conjugated antibody.
 Applications of Flow Cytometry
Clinical Applications
HIV monitoring
Leukemia/lymphoma immunophenotyping
Organ transplant monitoring
DNA analysis for tumor ploidy
Primary and secondary immunodeficiency
Hematopoietic reconstitution
Molecular Flow Cytometry
Multiplexing oligonucleotide assays
Measuring gene expression
In situ hybridization
Drug discovery

Research Applications
Multiplexing immunoassays
Multiparameter immunophenotyping
Measurement of intracellular cytokines
Signal transduction pathways
Cell cycle analysis
Measuring cellular function
 Immunostaining
Antibodies can be used to
visualize the presence of antigen
within the cell or the tissue.

In immunofluorescence the
fluorescence labeled antibody is
incubated with the cells or tissue
Figure: Antibody stained rat brain
and are visualized under the (including the ventricles and the CA3
region of the hippocampus) tissue.
fluorescence microscope.
 Cell Cycle analysis
Bromodeoxyuridine incorporates into DNA
in place of deoxythymidine during DNA
synthesis.

Propidium iodide intercalates into

DNA and acts as a cell cycle and
apoptosis indicator.
Fluorescence measured the FL2
(dye) shows cells bearing
amounts of DNA characteristic of
apoptotic cells, and cells in the
G1, S, and G2/M phases of the
cell cycle.
Thank You