Radio Immuno Assay

(RIA)
 The technique was introduced in 1960 by
Berson and Yalow as an assay for the
concentration of insulin in plasma.
It represented the first time that hormone
levels in the blood could be detected by an
in vitro assay.
 Principal
Based on competition between unlabelled antigen and
finite amount of corresponding labelled antigen for a
limited number of antibody binding sites in a fixed
amount of antiserum.
At equilibrium in the presence of an antigen excess
there will be both free antigen antigen bound to
antibody.
Under standard conditions the amount of labelled
antigen bound to the antibody will decrease as the
amount of unlabelled antigen in the sample increases.
 4Agg + 4 Ab 4Agg.Ab
4 Ag + 4 Agg + 4 Ab 2AgAbg + 2 AgAb +
2Agg + 2Ab
12 Ag + 4 Agg + 4 Ab Agg Ab + 3AgAb +
3Agg + 9 Ag
 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]
Preparation & Radiolabelling of the Ag, Ab
Antigens prepared by..
Synthesis of the molecule
Isolation from natural sources
Radiolabelling [Tagging procedure]
3 H 14 C 125 I are used as radioactive tags used for Ag
tagging
Tagging should NOT affect Antigenic specificity &
Antigenic activity !
Ab preparation: Antigen injected intradermally into rabbits
or guinea pigs antibody production
Antibodies recovered from the serum
Development of the Assay System
A crucial step is separation of unbound
antigens
This achieved by binding the antibodies to the
microtitre well surface [Solid phase RIA]
Antigens bound to the fixed antibodies
remain stuck to the inner surface
Decanting & washing the well removes
unbound antigens
Other techniques of separation:
Centrifugation
 Assay Procedure
Add known amounts of the test sample + labelled
antigen into the microtitre wells
Incubate allow the reaction to reach completion
Decant & wash contents of the well removes all
unbound antigens
Radioactivity remaining in the Microtitre wells
measured by a Counter [GM counter , Scintillation
counter etc]
Intensity of radioactivity is inversely correlated with
the conc of antigens in the test sample
Sensitive to very low conc of antigens
 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.
Advantages & Disadvantages of RIA
Advantages
Highly specific: Immune reactions are specific
High sensitivity: Immune reactions are sensitive
Disadvantages
Radiation hazards: Uses radiolabelled reagents
Requires specially trained persons
Labs require special license to handle radioactive
material
Requires special arrangements for
Requisition, storage of radioactive material
radioactive waste disposal.
 Advantages/Disadvantages
Advantages
Extremely sensitive and precise
Detects trace amounts of analytes small in size.
Disadvantages
Health hazard
Disposal problems
Short shelf life
Expensive equipment necessary
Enzyme immunoassays have largely
replaced radioimmunoassay.
 Applications
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).
Enzyme Linked Immunosorbent Assay

(ELISA)
 Enzyme Linked Immunosorbent Assay (ELISA)
Term Was Coined By Engvall and Pearlmann in 1971
Different Types
Sandwich (also called direct ELISA)
Indirect
Competitive
Similar To RIA, Detection based on
Enzyme catalysed reaction OR
Fluorescent probe
NOT radioactivity [great advantage!]
Can Be Used To Detect Both Antibody and Antigen
Very Sensitive, pg/mL
Relies on Monoclonal Abs
 Labeling
Principle technique
use of enzyme-labelled immunoglobulin to
detect antigens or antibodies
signals are developed by the action of
hydrolyzing enzyme on chromogenic substrate
optical density measured by micro-plate reader
Examples
Hepatitis A (Anti-HAV-IgM, anti-HAV IgG)
 Types of ELISA
1. Noncompetitive binding assay or
Sandwich method
1. Antigen measuring system [Titrewells coated
with antibodies ; Enzyme labelled antibodies]
2. Antibody measuring system/Indirect ELISA
[Titrewells coated with antigens ; Enzyme
labelled antiantibodies]
2. Competitive binding assay
[Titrewells
coated with antibodies ; Enzyme labelled
antigens]
 ELISA Plate

96 well plate
Made of plastic on which protein can be adsorbed
(bind) easily
Usually done overnight @ 4C
Special buffer used that will not denature Ab and
maximize binding
Blocking step ensures no empty spaces are left
Blocking reagent is often 10% FBS
 ELISA: Enzyme Choices
Enzyme labels should have high specific reactivity
Should be easily coupled to ligands & the labelled
complex must be stable
The reactivity should be retained after linking of
the enzyme to the antigen/antibody
The chosen enzymes should not be normally
present in the patient samples
Horse Radish Peroxidase
Alkaline Phosphatase
Glucose Oxidase
Urease
 Sandwich ELISA
Antigen measuring system
Titre wells coated with suitable antibody
Add patient sample containing the antigen
Incubate: till antigen antibody reaction is complete
Wash remove unbound antigen
Add antiantibody labelled with Enzyme
Incubate till antigen binds labelled antibody
Wash remove unbound labelled antibody
Add substrate ; incubate
Enzyme + Substrate Product measure colour
Colour proportional to antigen in patient sample
 Indirect ELISA
Antibody measuring system
Titre wells coated with suitable antigen
Add patient sample containing the antibody
Incubate: till antigen antibody reaction is complete
Wash remove unbound antibody
Add Antiantibody labelled with Enzyme
Incubate till labelled antiantibodies binds antigen-
antibody complex
Wash remove unbound labelled antiantibody
Add substrate ; incubate
Enzyme + Substrate Product measure colour
Colour proportional to antibody in patient sample
 Competitive ELISA
The labelled antigen competes for primary
antibody binding sites with the sample antigen
(unlabeled). The more antigen in the sample,
the less labelled antigen is retained in the well
and the weaker the signal).
 Competitive ELISA
Titrewells coated with antibodies
Known quantities of patient sample containing
antigen + antigen labelled with enzyme
Incubate: till antigen antibody reaction is complete
Wash remove unbound antigens
Add substrate ; incubate
Enzyme + Substrate Product measure
colour
Colour inversely related to antigen in patient
sample
 ELISA:
Performance, applications
Advantages
Automated, inexpensive
Sensitive: nanogram levels or lower
Small quantities required
Class specific antibodies measurable
Limitations
Expensive initial investment
Variable sensitivity / specificity of variable tests
Cross contamination
Time taken - 1 day
 Applications of Immunoassays
[RIA & ELISA]
Analysis of hormones, vitamins, metabolites,
diagnostic markers
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
 Labeling
Immuno-fluorescence
technique
Principle
Use fluorescein
isothiocyanate labeled-
immunoglobulin to detect
antigens or antibodies
according to test systems
Cell infected with Dengue virus
Requires a fluorescent
microscope
Examples
Herpes virus IgM
Dengue virus
Rabies virus
Scrub and murine typhus V. Cholerae
 Fluorescent Immunoassay
Markers
Fluorophores or fluorochromes
Ability to absorb energy and emit light
Two most commonly used:
Fluorescein green
Tetramethylrhodamine red
Tests may be qualitative or quantitative
 Fluorescent Immunoassay
Antibodies and bacteria are fixed on a glass-plate.
The surplus i.e. non-bounded antibodies are washed out,
antibody-bacteria-complexes ("sandwiches") remain.
The "sandwich" becomes visible by adding fluorescent
anti bovine immunoglobulin which can be seen as green
light in the fluorescence microscope.
 Labeling
Types of technique
immuno-fluorescence
Direct immuno-
fluorescence
Used to detect antigen
Indirect and
sandwich immuno-
fluorescence
Antigen detection
Antibody detection
 In direct immunostaining, the virus-specific antibody is
directly labeled with an indicator molecule, so as to permit its
visualization (the indicator molecule is usually either an
enzyme such as horseradish peroxidase or a fluorescent
molecule such as fluorescein). In indirect immunostaining,
the virus-specific antibody is detected using a labeled
secondary antibody directed against the first antibody (e.g., a
labeled anti-mouse antibody if the initial antibody was a
murine monoclonal antibody).
Fluorescent Antibody Techniques (Direct)
Fluorescent Antibody Techniques (Indirect)
 Virus antigen detection:
Immunofluorescence - direct
 Immuno-fluorescence:
Performance, applications
Advantages
Sensitive and specific
Can be used for discrepant analysis
Limitations
Expensive (Reagents and equipment)
Need good specimen
Cross reactivity/poor specificity
Non-specific immuno-fluorescence
Time taken
Few hours
Precipitation test :

Precipitation test can be performed in solution or

in semi- solid medium (agar).
Ab and soluble Ag interacting in aqueous solution form a
visible precipitate.
In this test antigen is in soluble form (solution).
Antibody cross -links antigen molecules to form
aggregates called as precipitin (precipitates) in the
zone of equivalence: optimal proportion of antigen
and antibody.
Ag-Ab complex occurs with in a minute where as
precipitation takes two days.
 Imunoprecipitation reaction
Used for qualitative and
quantitative detection of
antigens and antibodies:
phase one formation of
primary complexes with
low MW

Phase two
interconnection of Ag
and Ab to the three
dimensional network
(formation of insoluble
aggregates )
Zone of Equivalence
 Passive Immunodiffusion
Reactions of antigens and antibodies in agar
gel.
Migrate towards each other and where they meet
in optimal proportions form a precipitate.
Passive because they are allowed to react to
completion with no enhancements such as an
electrical charge applied.
Most widely used gels agar a agarose
Tests are performed by pouring molten agar
(agarose) onto glass slides
 Factors Affecting Rate of
Diffusion
Size of the particles.
Temperature
Gel viscosity and hydration
Interaction of reactants with gel
Types of immunodiffusion
Single radial immunodiffusion (mancini Mehod)
Double radial immunodiffusion (Ouchterlony
method).
Precipitatioin Agar.

Single radial immunodiffusion (Mancini Method):

- the process of diffusion of an antigen in an antibody-containing gel
- the process of diffusion of an antibody in an antigen-containing gel.
Antibody is incorporated into agar and antigen introduced
into the well.
As antigen diffuses into agar and precipitation rings
form depending on the concentration of the antigen.
The diameter of the ring directly proportional to concentration.
Create standard curve and read results.
Radial Immunodiffusion is used to measure IgG, IgM and
complement components.
Single Immunodiffusion

Zone of equivalence:
formation of large complexes
 Radial Immunodiffusion
Precipitin Rings
A B C
a b c
Standards Samples

Standard Curve
 RID Sources of Error
Over or under filling the well.
Spilling sample onto the outside of the well.
Nicking the well with the pipette tip.
Improper incubation time or temperature.
Incorrect measuring of the wells.
Double immunodiffusion (Ouchterlony method).

Antigen and antibody are placed in different

wells in agar and allowed to diffuse and
form precipitation lines at the points of
optimal concentrations.

This method is used to determine whether

antigens are related, identical or non
identical.
 Ouchterlony Gel Diffusion
Holes punched in agar.
Known antibody or antigen added to center well.
Known sample added to outer well.
Unknown sample added to outer well next to
unknown sample.
Wait for bands to form.
This is a QUALITATIVE technique, simply
determines the presence NOT the concentration.
Precipitation Test in agar:
 Precipitation and
immunodiffusion in gels
Double diffusion in two dimension

Similar precipitin Precipitin lines do Precipitin lines

lines not form a completely cross
complete cross
Double immunodiffusion
 Ouchterlony - Identity
Precipitation appears as a continuous line in the form of an arc
between the two outer wells and the center well. There are no
spurs at the angle and this type of reaction is termed a band of
identity.
 Ouchterlony Partial Identity
FIGURE 2:
If a solution with antigens X and Y is placed in well 1, a solution with
antigen X only is placed in well 2, and antiserum containing
antibodies specific for both X and Y is placed in well 3, a reaction
similar to that appearing in Fig. 2 will occur. Notice that there is a
spur reaction towards the XY well. This indicates that the two
antigenic materials in wells 1 and 2 are related, but that the material
in well 1 possesses an antigenic specificity not possessed by the
material in well 2. Such a reaction with spur formation indicates
partial identity
 Ouchterlony Non-Identity
If the material in wells 1 and 2 do not possess common
antigens and the antiserum in well 3 possesses specificities for
both materials, the reaction will appear as two crossed lines as
in Fig. 3
 Immunoelectrophoresis
Electrophoresis is a technique which
separates molecules using electrical current.
Immunodiffusion can be combined with
electrical current to speed things up.
Small molecules move faster than large.
For immunolectrophoresis antigen and
antibody migrate through gel faster.
 Immunoelectrophoresis
Two step double diffusion technique.
Electrophorese antigen.
Antiserum added to trough parallel to line of
separation.
Incubate overnight.
Diffusion occurs and bands of precipitate form.
Most often used as a screening test.
Immunoelectrophoresis
 Immunoelectrophoresis
Two-dimensional immunoelectrophoresis. Antigens are separated on
the basis of electrophoretic mobility. The second separation is run at
right angles to the first which drives the antigens into the antiserum-
containing gel to form precipitin peaks; the area under the peak is
related to the concentration of antigen.
 Precipitation and
immunodiffusion in gels
Immunoelectrophoresis
combines electrophoresis
Plasma separation, diffusion and
(mixture of
antigens) precipitation of proteins.
Electrophoresis
Ag mixture is first
electrophoresed, separate Ag
Antiserum
(mixture of
based on charge.
antibodies)
Cut the agar gel parallel to the
Imunodiffusion direction of electric field, add
antiserum.
Allowed to diffuse, a
precipitation arc will form.
Precipitation and immunodiffusion
in gels
 Rocket Immunoelectrophoresis
Adaptation of radial immunodiffusion
(RID).
Antibody incorporated (mixed) into the gel.
Antigen added to wells.
Apply electrical current and antigen will
move forward and will bind to antigen.
Dissolution and reformation occurs.
Height of precipitin band related to
concentration of antigen.
Much faster than RID.
 Rocket Immunoelectrophoresis
Antigen is electrophoresed into gel containing antibody.
The distance from the starting well to the front of the
rocket shaped arc is related to antigen concentration.
Electrophoresis Sources of Error
Applying current in wrong direction.
Incorrect buffer pH
Incorrect timing
Incorrect current applied.
Concentration of reactants must be
appropriate.
 Monoclonal Antibodies
Immunize Animal With Antigen
Multiple Clones Are Generated, Good For In Vivo
For Clinical Diagnosis, Research, One Clone That
Reacts To Single Epitope Is Preferred
Solution By Kohler and Milstein
Fuse A Myeloma Cell (Cancerous) With A Normal
Plasma Cells
Resulting Clones Can Be Cultured Indefinitely
Produces An Antibody Recognizing One Epitope
 Monoclonal Antibody Production
Method
Monoclonal Antibody Production technology was developed in
1975.
Monoclonal Antibody Production or mAb is produced by cell
lines or clones obtained from the immunized animals with the
substance to be studied. Cell lines are produced by fusing B
cells from the immunized animal with myeloma cells. To
produce the desired mAb, the cells must be grown in either of
two ways: by injection into the peritoneal cavity of a suitably
prepared mouse (the in vivo, or mouse ascites, method) or by in
vitro tissue culture.
The vitro tissue culture is the method used when the cells are
places in culture outside the mouse's body in a flask.
 Monoclonal Antibody
Applications
Diagnostic Tests
Abs are capable to detect tiny amouns (pg/mL) of molecules
Ex. Pregnancy hormones
Diagnostic Imaging
mAbs that recognize tumor antigens are radiolabeled with
iodine I-131
Immunotoxins
mAbs conjugated with toxins
mAbs To Clear Pathogens
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