CHAPTER-XII

IMMUNOCHEMICAL TECHNIQUES
Radioimmunoassay

Radioimmunoassay (RIA) is a very sensitive in vitro assay technique used to measure

concentrations of antigens (for example, hormone levels in the blood) by use of antibodies. As
such, it can be seen as the inverse of a radiobinding assay, which quantifies an antibody by use of
corresponding antigens.

Although the RIA technique is extremely sensitive and extremely specific, requiring specialized
equipment, it remains the least expensive method to perform such tests. It requires special
precautions and licensing, since radioactive substances are used. Today it has been supplanted by
the ELISA method, where the antigen-antibody reaction is measured using colorimetric signals
instead of a radioactive signal. However, because of its robustness, consistent results and low
price per test, RIA methods are again becoming popular. It is generally simpler to perform than a
bioassay.

The RAST test (radioallergosorbent test) is an example of radioimmunoassay. It is used to detect

the causative allergen for an allergy.

Method

To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently

by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. This radiolabeled
antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two
chemically 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 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 antigen remaining in the supernatant is measured using a gamma
counter. Using known standards, a binding curve can then be generated which allows the amount
of antigen in the patient's serum to be derived.
ELISA

.Enzyme-linked immunosorbent assay (ELISA), is a popular format of a "wet-lab" type

analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme
immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a liquid sample
or wet sample.

The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a
quality-control check in various industries. Attempting to detect (and quantify) the presence of
the antigen in the sample proceeds as follows: Antigens from the sample are attached to a
surface. Then, a further specific 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 in the substrate.
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 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 not 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, so are not easily reusable.

Principle

As a "wet lab" analytic biochemistry assay, ELISA involves detection of an "analyte" (i.e. the
specific substance whose presence is being quantitatively or qualitatively analyzed) in a liquid
sample by a method that continues to use liquid reagents during the "analysis" (i.e. controlled
sequence of biochemical reactions that will generate a signal which can be easily quantified and
interpreted as a measure of the amount of analyte in the sample) that stays liquid and remains
inside a reaction chamber or well needed to keep the reactants contained; It is opposed to "dry
lab" that can use dry strips - and even if the sample is liquid (e.g. a measured small drop), the
final detection step in "dry" analysis involves reading of a dried strip by methods such as
reflectometry and does not need a reaction containment chamber to prevent spillover or mixing
between samples.

As a heterogenous assay, ELISA separates some component of the analytical reaction mixture by
adsorbing certain components onto a solid phase which is physically immobilized. In ELISA, a
liquid sample is added onto a stationary solid phase with special binding properties and is
followed by multiple liquid reagents that are sequentially added, incubated and washed followed
by some optical change (e.g. color development by the product of an enzymatic reaction) in the
final liquid in the well from which the quantity of the analyte is measured. The qualitative
"reading" usually based on detection of intensity of transmitted light by spectrophotometry,
which involves quantitation of transmission of some specific wavelength of light through the
liquid (as well as the transparent bottom of the well in the multiple-well plate format). The
sensitivity of detection depends on amplification of the signal during the analytic reactions. Since
enzyme reactions arre very welll known am mplificationn processes, the signal is generateed by
enzymes which are linked to the t detectioon reagents in fixed prroportions too allow acccurate
quantificcation - thus the name "ennzyme linkeed".

The anallyte is also called the ligand becauuse it will specifically bind b or ligaate to a deteection
reagent, thus
t ELISA falls under the t bigger caategory of liigand bindinng assays. Thhe ligand-speecific
binding reagent
r is "immobilizedd", i.e., usuallly coated annd dried ontoo the transparent bottom m and
sometimees also side wall of a weell (the statioonary "solidd phase'/"soliid substrate"" here as oppposed
to solid microparticcle/beads thaat can be washed w awaay), which is usually constructed
c as a
multiple--well plate known as the "ELIS SA plate". Conventionnally, like other form ms of
immunoaassays, the sp pecificity off antigen-anttibody type reaction
r is ussed because it is easy to raise
an antiboody specificaally against an a antigen inn bulk as a reagent. Alteernatively, iff the analyte itself
is an antiibody, its tarrget antigen can
c be used as the bindinng reagent.

Types

"Indirecct" ELISA
Direct EL
LISA diagraam

The stepss of "indirecct" ELISA foollows the mechanism beelow:-

• A buffered so olution of thhe antigen too be tested for

f is addedd to each weell of a micrrotiter
plate, where iti is given tim
me to adheree to the plasttic through charge
c interaactions.
• A solution off nonreacting protein, suuch as bovinne serum albbumin or caasein, is addded to
block any plaastic surface in the well that
t remains uncoated byy the antigenn.
• T primary antibody is added, whiich binds specifically too the test anttigen coatinng the
The
w
well. This prrimary antibbody could also be in the serum of a donor to be tested for
reeactivity tow
wards the anttigen.
• A secondary antibody iss added, whiich will bind the primaary antibodyy. This seconndary
anntibody ofteen has an enzzyme attacheed to it, whiich has a neggligible effect on the binnding
prroperties of the antibodyy.
• A substrate forf this enzzyme is thenn added. Offten, this suubstrate chaanges color upon
reeaction withh the enzymee. The color change shows the secondary antiboody has bouund to
prrimary antib body, whichh strongly im mplies the doonor has hadd an immunne reaction to t the
teest antigen. This
T can be helpful
h in a clinical
c setting, and in reesearch.
• T higher th
The he concentraation of the primary anttibody presennt in the serrum, the stroonger
thhe color chaange. Often, a spectrom meter is useed to give quantitative
q values for color
sttrength.

The enzyyme acts as an amplifierr; even if onnly few enzyyme-linked antibodies

a reemain boundd, the
enzyme molecules
m will
w produce many signaal moleculess. Within coommon-sense limitationss, the
enzyme canc go on prroducing color indefinittely, but the more primaary antibodyy is present in i the
donor seerum, the mo ore secondaary antibodyy + enzyme will bind, and a the fastter the colorr will
develop. A major dissadvantage of o the indirecct ELISA is the method of antigen immobilizati
i ion is
not speciific; when serum
s is useed as the souurce of test antigen, all proteins in the sample may
stick to the microtiteer plate well,, so small cooncentrations of analyte in serum muust competee with
other seruum proteins when bindinng to the weell surface. The
T sandwichh or direct ELISA
E proviides a
solution to this probllem, by usinng a "capturee" antibody specific for the test antiigen to pull it out
of the serrum's molecu ular mixturee.

ELISA may m be run in a qualitattive or quanntitative form mat. Qualitattive results provide a siimple
positive oro negative result (yes or
o no) for a sample. Thee cutoff betw ween positivve and negatiive is
determinned by the an nalyst and may
m be statistical. Two orr three times the standardd deviation (error
(
inherent in a test) iss often usedd to distinguuish positivee from negattive sampless. In quantittative
ELISA, the optical density (OD D) of the sample
s is coompared to a standard curve, which is
typically a serial dilu
ution of a knnown-concenntration soluttion of the taarget molecuule. For exammple,
if a test sample
s returnns an OD off 1.0, the poiint on the staandard curvee that gave OD
O = 1.0 muust be
of the samme analyte concentration
c n as the sam
mple.
Sandwicch ELISA

A sandwwich ELISA A. (1) Plate is

i coated witth a capturee antibody; (2)
( sample iss added, andd any
antigen present
p bindss to capture antibody; (33) detecting antibody is added, and binds to anttigen;
(4) enzymme-linked seecondary anttibody is addded, and binnds to detectting antibodyy; (5) substrate is
added, annd is convertted by enzymme to detectaable form.

A less-coommon variaant of this teechnique, a "sandwich" ELISA, is used to detect sample anttigen.
The stepss are:

1. A surface is prepared
p to which
w a knowwn quantity of capture antibody is bound.
2. A nonspeciific binding sites on the surface are blocked.
Any b
3. T antigen-ccontaining saample is appplied to the plate.
The p
4. T plate is washed
The w to rem
move unbouund antigen.
5. A specific an ntibody is addded, and binnds to antigeen (hence thhe 'sandwich': the Ag is stuck
between two antibodies)
6. E
Enzyme-linke ed secondarry antibodiess are applied as detection antibodiees that also bind
sppecifically to
o the antiboddy's Fc regioon (nonspeciific).
7. T plate is washed
The w to rem
move the unnbound antibbody-enzymee conjugates.
8. A chemical is added too be convertted by the enzyme intto a color or o fluorescent or
ellectrochemiccal signal.
9. T absorben
The ncy or fluoreescence or ellectrochemiccal signal (e.g., current) of the plate wells
iss measured to determine the presence and quantiity of antigenn.

The image to the riight includees the use of o a secondaary antibodyy conjugatedd to an enzzyme,
though, in
i the techniical sense, thhis is not neecessary if the
t primary antibody is conjugated to an
enzyme. However, use of a seecondary-anttibody conjugate avoidds the expennsive process of
creating enzyme-link ked antiboddies for everry antigen one o might want w to deteect. By usinng an
enzyme-llinked antibody that binnds the Fc region
r of othher antibodies, this samme enzyme-liinked
antibody can be used d in a varietyy of situationns. Without the first layeer of "capturre" antibodyy, any
proteins in the samplle (includingg serum protteins) may competitively
c y adsorb to the
t plate surrface,
loweringg the quantity
y of antigen immobilized. Use of thhe purified sppecific antibbody to attacch the
antigen to
t the plasticc eliminates a need to purify
p the anntigen from complicatedd mixtures before
b
the meassurement, simmplifying thhe assay, andd increasing the specificcity and the sensitivity of o the
assay.
Competitive ELISA

A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat
different from the first two examples:

1. Unlabeled antibody is incubated in the presence of its antigen (sample).

2. These bound antibody/antigen complexes are then added to an antigen-coated well.
3. The plate is washed, so unbound antibody is removed. (The more antigen in the sample,
the less antibody will be able to bind to the antigen in the well, hence "competition".)
4. The secondary antibody, specific to the primary antibody, is added. This second antibody
is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
6. The reaction is stopped to prevent eventual saturation of the signal.

Some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked
antibody. The labeled antigen competes for primary antibody binding sites with the sample
antigen (unlabeled). The more antigen in the sample, the less labeled antigen is retained in the
well and the weaker the signal.

Commonly, the antigen is not first positioned in the well.

For the detection of HIV antibodies, the wells of microtiter plate are coated with the HIV
antigen. Two specific antibodies are used, one conjugated with enzyme and the other present in
serum (if serum is positive for the antibody). Competition occurs between the two antibodies for
the same antigen. Sera to be tested are added to these wells and incubated at 37°C, and then
washed. If antibodies are present, the antigen-antibody reaction occurs. No antigen is left for the
enzyme-labelled specific HIV antibodies. These antibodies remain free upon addition and are
washed off during washing. Substrate is added, but there is no enzyme to act on it, so positive
result shows no color change.

Multiple and portable ELISA

A new technique (EP 1 499 894 B1 in EPO Bulletin 25.02.209 N. 2009/09; USPTO 7510687 in
USPTO Bulletin 31.03.2009; ZL 03810029.0 in SIPO PRC Bulletin 08.04.2009) uses a solid
phase made up of an immunosorbent polystyrene rod with eight to 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 chromogens) are carried out by dipping the
ogives in microwells of standard microplates filled with reagents.

The advantages of this technique are:

1. The ogives can each be sensitized to a different reagent, allowing the simultaneous
detection of different antibodies and/or different antigens for multiple-target assays.
2. The sample volume can be increased to improve the test sensitivity in clinical (blood,
saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples.
3. One ogive is left unsensitized to measure the nonspecific reactions of the sample.
 4. The use of laboratory supplies for dispensing sample aliquots, washing solution and
reagents in microwells is not required, facilitating the development of ready-to-use lab
kits and on-site testing.

Applications
Human anti-IgG, double antibody sandwich ELISA

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 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. ELISA can
also be used in toxicology as a rapid presumptive screen for certain classes of drugs.

Enzyme-linked immunosorbent assay plate

The ELISA was the first screening test widely used for HIV because of its high sensitivity. In an
ELISA, a person's serum is diluted 400 times and applied to a plate to which HIV antigens are
attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The
plate is then washed to remove all other components of the serum. A specially prepared
"secondary antibody" — an antibody that binds to other antibodies — is then applied to the plate,
followed by another wash. This secondary antibody is chemically linked in advance to an
enzyme.

Thus, the plate will contain enzyme in proportion to the amount of secondary antibody bound to
the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in
color or fluorescence. ELISA results are reported as a number; the most controversial aspect of
this test is determining the "cut-off" point between a positive and a negative result.

A cut-off point may be determined by comparing it with a known standard. If an ELISA test is
used for drug screening at workplace, a cut-off concentration, 50 ng/ml, for example, is
established, and a sample containing the standard concentration of analyte will be prepared.
Unknowns that generate a stronger signal than the known sample are "positive." Those that
generate weaker signal are "negative".ELISA tests also are used as in in vitro diagnostics in
medical laboratories.

K.ANITA PRIYADHARSHINI

LECTURER

DEPT.OFPHARMACEUTICAL CHEMISTRY

SRM COLLEGE OF PHARMACY