ELISA, RIA &
Immunofluorescence
ELISA Definition:
• The enzyme-linked immunosorbent assay (ELISA) is a
common laboratory technique which is used to measure
the concentration of an analyte (usually antibodies or
antigens) in solution.
• The term ELISA was first used by Engvall & Perlma in 1971.
BASIC PRINCIPLE OF ELISA
 Based on Basic Immunology Response
 Lock and Key Concept:
1) Antigen (key)
2) Antibody (lock):
–Key fits into the lock
 Enzyme conjugate substrates
• Bound to a secondary antibody that binds with the
antibody-antigen complex.
Basic Steps Of Enzyme-Linked
Immunosorbant Assay
Indirect
Types of ELISA:
(on the basis of procedure)
Direct
Types
NonCompetitive
Indirect
Competitive
Sandwich
Non-Competitive:
1) Direct ELISA:
• It uses a primary labeled anti-body that react directly
with the antigen.
• It can be performed with the antigen that is directly
immobilized on assay plate.
• Not widely used but common for immuno-histochemical
staining of cells & tissues.
Non-Competitive:
2) Indirect ELISA:
• It utilizes a primary un-labeled antibody in conjunction
with a labeled secondary antibody.
• Secondary antibody has specificity for primary antibody.
Non-Competitive:
3) Sandwich ELISA:
• Antigens like Tumor markers, hormones, serum proteins
may be determined.
• Antigens in the sample bind with the capture antibody &
become immobilized.
• The antibody of the enzyme conjugate bind with the
immobilized antigen to form a sandwich of Ab-Ag-Ab/
enzyme bound to microwell.
Competitive:
• Antibody coated microwell.
• Serum antigen & labeled antigen added together .... Competition
• Ab-Ag enzyme complex bound is inversely related to the conc. of
antigen present in sample.
• Increased serum antigen results in reduced binding of Agenzyme conjugate with the antibody producing less enzyme
activity & (yellow) color formation.
• Used to determine small molecules like T₃ , T₄ & Progesterone.
to detect Ab (HIV, HCV)
to detect Ag ( Tumor Markers, Hormones )
to detect Ag ( Free Testosterone)
Comparison between Indirect Sandwich & Competitive ELISA
APPLICATIONS OF ELISA
1- Hormones
7- Vaccine Quality Control
2- Proteins
8- FOR GMO (Genetically modified
organism)
3- Infectious Agent ( Viral, Bacterial,
Parasitic, Fungal )
9- For Rapid Test
4- Drug Markers
10- IgG, IgM, IgA
5- Tumor Markers
11- In New Born Screening
6- Serum Proteins
12- In Clinical Research
Radioimmunoassay (RIA)
•Involves the separation of a protein (from a mixture)
using the specificity of antibody - antigen binding and
quantify it using radioactivity
•The technique was introduced in 1960 by Berson
and Yalow as an assay for the concentration of
insulin in plasma.
•Here radioactive materials are not administered
to the individual but are used as reagents.
14
The technique of radioimmunoassay has
revolutionized research and clinical practice
in many areas, e.g.,
✓blood banking
✓diagnosis of
allergies
✓endocrinology
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Principle of Radioimmunoassay
Principle:
Uses an immune reaction [Antigen –
Antibody reaction] to estimate a ligand
Ag + Ag* + Ab
AgAb + Ag*Ab + Ag + Ab*
◦ Unbound Ag* and Ag washed out
◦ Radioactivity of bound residue measured
◦ Ligand conc. is inversely related to
radioactivity
[Ag : ligand to be measured ; Ag* radiolabelled
ligand]
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P* +
Radioactive tag
PQ
Q
+
P
P*Q
Analyte
Binding agent
Free
Bound
17
✓Amixture is prepared of
▪ radioactive antigen
▪ antibodies against that antigen.
✓Known amounts of unlabeled ("cold") antigen are added
to samples of the mixture. These compete for the binding
sites of the antibodies.
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✓At increasing concentrations of unlabeled antigen,
an increasing amount of radioactive antigen is
displaced from the antibody molecules.
✓The antibody-bound antigen is separated from
the free antigen in the supernatant fluid, and
✓ The radioactivity of each is measured.
19
20
Applications of RIA
❑
Analysis of hormones,
vitamins,metabolites, diagnosticmarkers
◦ Eg. ACTH, FSH, T3, T4, Glucagon, Insulin,
Testosterone, vitamin B12, prostaglandins,
glucocorticoids,
❑Therapeutic
drug monitoring:
◦ Barbiturates, morphine, digoxin,
❑
Diagnostic procedures for detecting infection
◦ HIV, Hepatitis A, B etc
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RIA
Sensitivity
Nanomolar to
picomolar
More
EIA
Millimolar
Cost
Time duration More
Tedious
Ease of
handling
More
Radiation
hazards
Less
Disposal
Care has to be
taken
Complex
Easy
Based on the
measurement of
radioactivity
Based on
measurement of28
enzymatic
Equipment
Mechanism
Less
Easy
No
Less
Immunofluorescence
• Immunofluorescence is a technique allowing the
visualization of a specific antigen by binding a
specific antibody chemically conjugated with a
fluorescent dye such as fluorescein isothiocyanate
(FITC).
• The specific antibodies are labeled with a
compound (FITC) that makes them glow an applegreen color when observed microscopically under
ultraviolet light.
Fluorescence
• Fluorescence is the property of
certain molecules to absorb
light at one wave length and
emit light at longer wave length
when it is illuminated by
light of a different
wavelength.
• The fluorescence can be
visualized using fluorescence
microscopy. The IF technique
allows for a visualization of the
presence as well as the
distribution of target molecules
in a sample.
Immunofluorescence staining
 Direct immunofluorescence: Staining in which
the primary antibody is labeled with
fluorescence dye.
 Indirect immunofluorescence: Staining in which
a secondary antibody labeled with
fluorochrome is used to recognize a primary
antibody.
Direct immunofluorescence
•
Direct immunofluorescence uses a single
antibody that is chemically linked to a
fluorophore. The antibody recognizes the
target molecule and binds to it, and the
fluorophore it carries can be detected via
microscopy.
Advantages of direct immunofluorescence
• This technique has several advantages over
indirect immunofluorescence because of the
direct conjugation of the antibody to the
fluorophore. This reduces the number of steps
in the staining procedure making the process
faster and can reduce background signal by
avoiding some issues with antibody crossreactivity.
Indirect immunofluorescence
•
Indirect immunofluorescence uses two
antibodies; the unlabelled primary antibody
specifically binds the target molecule, and the
secondary antibody, which carries the
fluorophore, recognises the primary antibody
and binds to it.
Advantages of indirect immunofluorescence
• Gives an amplification effect - more label per
molecule of target protein.
• Requires only one labeled antibody to identify
many proteins- Same labeled secondary antibody
can be used to bind to many different proteins.
A different primary antibody is used for each
target protein. Variable part of primary
antibody binds to specific part of target
protein.
• The secondary antibody binds to the constant
part of the primary antibody. Therefore a
sample of the same batch of secondary
antibody can bind to many different primary
antibodies
•
Applications
• Immunofluorescence can be used on tissue
sections, cultured cell lines, or individual cells,
and may be used to analyse the distribution
of proteins and small biological and nonbiological molecules.
• Immunofluorescence can be used in
combination with other, non-antibody
methods of fluorescent staining, for example,
use of DAPI, (4',6-diamidino-2-phenylindole)
is a fluorescent stain that binds strongly to A-T
rich regions in DNA, to label DNA
•
The technique has a number of different
biological applications including evaluation of
cells in suspension, cultured cells, tissue,
beads.
• It also play a key role in the diagnosis of
autoimmune disorder.
Limitation
•
•
•
•
Quality and concentration of the antibody.
Proper handling of the specimen.
Choice of secondary antibodies.
Fluorophores undergoes photobleaching as
they are exposed to light