Radioimmunoassay and related immunoassay techniques

radioimmunoassay and related immunoassay techniques

1. Pharmaceutical analysis-III
Radioimmunoassay and related immunoassay
techniques.
By: - Drx Jayesh.M.Rajput
Points:-
1. Radioimmunoassay
2. Elisa techniques,
Theory, instrumentation and applications.

1. Radioimmunoassay
A radioimmunoassay (RIA) is an immunoassay that uses radiolabeled molecules in a stepwise formation
of immune complexes. A RIA is a very sensitive in vitro assay technique used to measure concentrations
of substances, usually measuring antigen concentrations (for example, hormone levels in blood) by use of
antibodies.
Although the RIA technique is extremely sensitive and extremely specific, requiring specialized
equipment, it remains among the least expensive methods to perform such measurements. It requires
special precautions and licensing, since radioactive substances are used.
In contrast, an immunoradiometric assay (IRMA) is an immunoassay that uses radiolabeled molecules but
in an immediate rather than stepwise way.
A radioallergosorbent test (RAST) is an example of radioimmunoassay. It is used to detect the causative
allergen for an allergy.
Method
Classically, to perform a radioimmunoassay, a known quantity of an antigen is made radioactive,
frequently by labeling it with gamma-radioactive isotopes of iodine, such as 125-I, attached to
tyrosine. This radiolabeled antigen is then mixed with a known amount of antibody for that
antigen, and as a result, the two specifically bind to one another. Then, a sample of serum from a
patient containing an unknown quantity of that same antigen is added. This causes the unlabeled
(or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody

2. binding sites. As the concentration of "cold" antigen is increased, more of it binds to the
antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound
radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the
unbound ones, and the radioactivity of the free(unbound) antigen remaining in the supernatant is
measured using a gamma counter.
This method can be used for any biological molecule in principle and is not restricted to serum
antigens, nor is it required to use the indirect method of measuring the free antigen instead of
directly measuring the captured antigen. For example, if it is undesirable or not possible to
radiolabel the antigen or target molecule of interest, a RIA can be done if two different
antibodies that recognize the target are available and the target is large enough (e.g., a protein) to
present multiple epitopes to the antibodies. One antibody would be radiolabeled as above while
the other would remain unmodified. The RIA would begin with the "cold" unlabeled antibody
being allowed to interact and bind to the target molecule in solution. Preferably, this unlabeled
antibody is immobilized in some way, such as coupled to an agarose bead, coated to a surface,
etc. Next, the "hot" radiolabeled antibody is allowed to interact with the first antibody-target
molecule complex. After extensive washing, the direct amount of radioactive antibody bound is
measured and the amount of target molecule quantified by comparing it to a reference amount
assayed at the same time. This method is similar in principle to the non-radioactive sandwich
ELISA method.

2. Elisa technique.
The enzyme-linked immunosorbent assay (ELISA) (/ɪˈlaɪzə/, /ˌiːˈlaɪzə/) is a commonly used
analytical biochemistry assay, first described by Engvall and Perlmann in 1972.
[1]
The assay uses
a solid-phase enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a
protein) in a liquid sample using antibodies directed against the protein to be measured. ELISA
has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a
quality control check in various industries.

Theory:-

In the most simple form of an ELISA, antigens from the sample are attached to a surface. Then, a
matching antibody is applied over the surface so it can bind to the antigen. This antibody is
linked to an enzyme, and in the final step, a substance containing the enzyme's substrate is
added. The subsequent reaction produces a detectable signal, most commonly a color change.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The
sample with an unknown amount of antigen is immobilized on a solid support (usually a
polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically
(via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the
antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The
detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary

3. antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is
typically washed with a mild detergent solution to remove any proteins or antibodies that are
non-specifically bound. After the final wash step, the plate is developed by adding an enzymatic
substrate to produce a visible signal, which indicates the quantity of antigen in the sample.
Of note, ELISA can perform other forms of ligand binding assays instead of strictly "immuno"
assays, though the name carried the original "immuno" because of the common use and history
of development of this method. The technique essentially requires any ligating reagent that can
be immobilized on the solid phase along with a detection reagent that will bind specifically and
use an enzyme to generate a signal that can be properly quantified. In between the washes, only
the ligand and its specific binding counterparts remain specifically bound or "immunosorbed" by
antigen-antibody interactions to the solid phase, while the nonspecific or unbound components
are washed away. Unlike other spectrophotometric wet lab assay formats where the same
reaction well (e.g., a cuvette) can be reused after washing, the ELISA plates have the reaction
products immunosorbed on the solid phase, which is part of the plate, and so are not easily
reusable.
Types:-
1. Direct Elisa

In direct ELISA, only an enzyme-labeled primary antibody is used, meaning
that secondary antibodies are not needed. The enzyme-labeled primary
antibody "directly" binds to the target (antigen) that is immobilized to the
plate (solid surface). Next, the enzyme linked to the primary antibody
reacts with its substrate to produce a visible signal that can be measured. In
this way, the antigen of interest is detected.


2. Indirect Elisa

4. In indirect ELISA, both a primary antibody and a secondary antibody are used. But in this case, the primary
antibody is not labeled with an enzyme. Instead, the secondary antibody is labeled with an enzyme.
The primary antibody binds to the antigen immobilized to the plate, and then the enzyme-labeled secondary
antibody binds to the primary antibody. Finally, the enzyme linked to the secondary antibody reacts with its
substrate to produce a visible signal that can be measured.

3. Sandwich Elisa

In direct and indirect ELISA, it is the antigen that is immobilized to the plate. In sandwich ELISA, however, it is the
antibody that is immobilized to the plate, and this antibody is called capture antibody. In addition to capture
antibody, sandwich ELISA also involves the use of detection antibodies, which generally include the unlabeled
primary detection antibody and the enzyme-labeled secondary detection antibody.
Firstly, the antigen of interest binds to the capture antibody immobilized to the plate. Secondly, the primary
detection antibody binds to the antigen. Thirdly, the secondary detection antibody binds to the primary detection
antibody, and then the enzyme reacts with its substrate to produce a visible signal that can be measured.


4. Competitive Elisa

5. Compared with the three ELISA types above, competitive ELISA is relatively complex because it
involves the use of inhibitor antigen, so competitive ELISA is also known as inhibition ELISA. In fact,
each of the three formats, direct, indirect, and sandwich, can be adapted to the competitive format. In
competitive ELISA, the inhibitor antigen and the antigen of interest compete for binding to the primary
antibody. Here is a procedure of competitive ELISA:
Firstly, the unlabeled primary antibody is incubated with the sample containing the antigen of interest,
leading to the formation of antigen-antibody complex (Ag-Ab). In this step, the antibody is excessive
compared with the antigen, so there are free antibodies left.
Secondly, the Ag-Ab mixture is added to the plate coated with inhibitor antigen that can also bind to the
primary antibody. The free primary antibody in the mixture binds to the inhibitor antigen on the plate,
while the Ag-Ab complexes in the mixture do not and are therefore washed off.
Thirdly, the enzyme-labeled secondary antibody is added to the plate and binds to the primary antibody
bound to the inhibitor antigen on the plate.
Finally, a substrate is added to react with the enzyme and emit a visible signal for detection.
Through this procedure, you may find that the final signal is inversely associated with the amount of the
antigen of interest in the sample, meaning that the more antigen in the sample, the weaker the final signal.
This is because primary antibodies bound to sample antigen will be washed off, while free primary
antibodies left will be captured by inhibitor antigen immobilized to the plate and be measured by an
enzymatic reaction.
Competitive ELISA described here is based on antibody capture, in which the plate is coated with
antigen. There is another type of competitive ELISA that is based on antigen capture, in which the plate is
coated with unlabeled antibody. Furthermore, competitive ELISA generally uses a labeled antibody for
detection, but sometimes it uses labeled antigen instead of a labeled antibody.
Instrumentation
1. Elisa plate. 2. Positive control, 3.Negative control, 4.Dilution buffer,
5. Conjugate, 6. TMB substrate, 7. Stock solution.

7. Applications
 detection of Mycobacterium antibodies in tuberculosis
 detection of rotavirus in feces
 detection of hepatitis B markers in serum
 detection of hepatitis C markers in serum
 detection of enterotoxin of E. coli in feces
 detection of HIV antibodies in blood samples
Because the ELISA can be performed to evaluate either the presence of antigen or the presence of
antibody in a sample, it is a useful tool for determining serum antibody concentrations (such as with the
HIV test
[25]
or West Nile virus). It has also found applications in the food industry in detecting potential
food allergens, such as milk, peanuts, walnuts, almonds, and eggs
[26]
and as serological blood test for
coeliac disease.
[27][28]
ELISA can also be used in toxicology as a rapid presumptive screen for certain
classes of drugs.
A new Elisa technique (reverse Elisa)
A new technique uses a solid phase made up of an immunosorbent polystyrene rod with a 4-12 protruding
ogives. The entire device is immersed in a test tube containing the collected sample and the following
steps (washing, incubation in conjugate and incubation in chromigeneous) are carried out by dipping the
ogives in microwells of standard microplated prefilled with reagents.
Advantages of this technique
1. The sample volume can be increased to improve the test sensitivity in clinical, food and environmental
samples
2. The use of laboratory supplies for dispensing samples aliquots, washing solution and reagents in
microwells is not required, facilitating ready to use lab kits and on site kits.



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