ELISA and RIA

Guided by: Presented By :
Dr . Mahesh Kumar Kataria Nitish Chugh
Professor and Head, Department of Pharmaceutics M.Pharmacy (Pharmaceutics)
SETH G. L. BIHANI S. D. COLLEGE OF TECHNICAL EDUCATION,
SRI GANGANAGAR (RAJ.)
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 1

 ELISA (enzyme-linked immunosorbent assay) is a plate-based assay
technique designed for detecting and quantifying peptides, proteins, antibodies
and hormones. In an ELISA, an antigen must be immobilized to a solid surface
and then complexed with an antibody that is linked to an enzyme. Detection is
accomplished by assessing the conjugated enzyme activity via incubation with a
substrate to produce a measureable product. The most crucial element of the
detection strategy is a highly specific antibody-antigen interaction.
 ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which
will passively bind antibodies and proteins. It is this binding and immobilization
of reagents that makes ELISAs so easy to design and perform. Having the
reactants of the ELISA immobilized to the microplate surface makes it easy to
separate bound from non-bound material during the assay. This ability to wash
away nonspecifically bound materials makes the ELISA a powerful tool for
measuring specific analytes within a crude preparation.
 ELISA is an antigen antibody reaction. In 1971, ELISA was introduced by Peter
Perlmann and Eva Engvall at Stockholm University in Sweden. It is a common
laboratory technique which is usually used to measure the concentration of
antibodies or antigens in blood. An enzyme conjugated with an antibody reacts
with colorless substrate to generate a colored product. Such substrate is called
chromogenic substrate. A number of enzymes have been used for ELISA such as
alkaline phosphatase, horse radish peroxidase and beta galactosidase. Specific
substrate such as ortho-phenyldiamine dihydrochloride (for peroxidase),
paranitrophenyl phosphate (for alkaline phosphatase) are used which are
hydrolysed by above enzymes to give colored end product.

ELISAs are typically performed in 96-well polystyrene plates. The
serum is incubated in a well, and each well contains a different
serum. A positive control serum and a negative control serum
would be included among the 96 samples being tested.
Antibodies or antigens present in serum are captured by
corresponding antigen or antibody coated on to the solid
surface. After some time, the plate is washed to remove serum
and unbound antibodies or antigens with a series of wash
buffer. To detect the bound antibodies or antigens, a secondary
antibodies that are attached to an enzyme such as peroxidase
or alkaline phosphatase are added to each well.
After an incubation period, the unbound secondary antibodies are
washed off. When a suitable substrate is added, the enzyme
reacts with it to produce a color. This color produced is
measurable as a function or quantity of antigens or antibodies
present in the given sample. The intensity of color/ optical
density is measured at 450nm. The intensity of the color gives
an indication of the amount of antigen or antibody.

 Indirect ELISA
 Sandwich ELISA
 Competitive ELISA
 Direct ELISA

 Antibody can be detected or quantitatively determined by indirect
ELISA. In this technique, antigen is coated on the microtiter well.
Serum or some other sample containing primary antibody is added
to the microtiter well and allowed to react with the coated antigen.
Any free primary antibody is washed away and the bound antibody
to the antigen is detected by adding an enzyme conjugated
secondary antibody that binds to the primary antibody.

 Antigen can be detected by sandwich ELISA. In this technique,
antibody is coated on the microtiter well. A sample containing
antigen is added to the well and allowed to react with the
antibody attached to the well, forming antigen-antibody
complex. After the well is washed, a second enzyme-linked
antibody specific for a different epitope on the antigen is
added and allowed to react with the bound antigen. Then
after unbound secondary antibody is removed by washing.
Finally substrate is added to the plate which is hydrolyzed by
enzyme to form colored products.

 In this test, antibody is first incubated in solution with a
sample containing antigen. The antigen-antibody mixture is
then added to the microtitre well which is coated with
antigen. The more the antigen present in the sample, the less
free antibody will be available to bind to the antigen-coated
well. After the well is washed, enzyme conjugated secondary
antibody specific for isotype of the primary antibody is added
to determine the amount of primary antibody bound to the
well. The higher the concentration of antigen in the sample,
the lower the absorbance.

 For direct detection, an antigen coated to a multi-
well plate is detected by an antibody that has been
directly conjugated to an enzyme. This detection
method is a good option if there is no
commercially available ELISA kits for your target
protein

 Presence of antigen or the presence of
antibody in a sample can be evaluated.
 Determination of serum antibody
concentrations in a virus test.
 Used in food industry when detecting
potential food allergens.
 Applied in disease outbreaks- tracking the
spread of disease e.g. HIV, bird flu, common,
colds, cholera, STD etc.

 RIA or Radioimmunoassay is an in vitro assay that measures
the presence of an antigen with very high sensitivity. Basically
any biological substance for which a specific antibody exists
can be measured, even in minute concentrations. RIA has
been the first immunoassay technique developed to analyze
nanomolar and picomolar concentrations of hormones in
biological fluids.

Radioimmunoassay is based upon the competition between
labeled and unlabeled antigen for specific antibody sites,
forming antigen-antibody complexes. This reaction is
described by the expression see journal for formula. At
equilibirum, the radioactive complex (bound) is separated from
the radioactive antigen (free). The B/F ratio is dependent upon
the amount of nonradioactive antigen. Antigen concentration in
unknown samples is determined by comparing the B/F ratio to
the B/F ratios obtained by incubating varying amounts of
known nonradioactive antigen with the same amount of
antibody as in the unknown sample under similar assay
conditions. Sensitivity of the order of 10-12 moles/liter may be
achieved through the preparation and use of a labeled antigen
of high specific activity and the production and selection of
antisera with appropriately high affinity constants. Specificity is
dependent upon the ability of the antiserum to recognize
subtle structural features of the antigen molecule. The ability to
conveniently assay large numbers of samples with good
precision has led to the application of this technique to
quantitate substances (such as steroids) already measurable
but by more cumbersome methods.

 The target antigen is labeled radioactively and bound to its specific
antibodies; a limited and known amount of the specific antibody has to be
added. A sample, for example a blood-serum, is then added in order to
initiate a competitive reaction of the labeled antigens from the preparation,
and the unlabeled antigens from the serum-sample, with the specific
antibodies. The competition for the antibodies will release a certain amount
of labeled antigen. This amount is proportional to the ratio of labeled to
unlabeled antigen. A binding curve can then be generated which allows the
amount of antigen in the patient’s serum to be derived.

 As an example of how this technique works,
let’s apply it to insulin. To measure insulin,
the first step is to mix known amounts of
radioisotope-tagged insulin and antibodies.
These combine chemically. Next, a small
amount of the patient’s blood is added. The
insulin contained in the blood displaces some
of the tagged insulin. The free-tagged insulin
is then measured with isotope detectors and
the patient’s insulin level is calculated

 A plot of the distribution of radioactivity as a
function of the amount of unlabeled antigen present
is known as the standard or dose response curve.
Standard curves can be plotted in a variety of ways.
The most commonly used response curves are the
bound-free (B/F), free-bound (F/B), fraction bound
(B) of occasionally the percent bound B/Bo ratios.
The dose can be plotted on either the arithmatic or
logarithmic scale. To obtain response curves that
are essentially linear over a large part of the antigen
concentration, the logit function defined as follows
has been used: Logit(y) = where y = either B/Bo or
(B/F)/(Bo/Fo) When logit (y) is plotted against the
log concentration of the antigen, a linear dose-
response curve results for most of the assay system.

 Radioimmunoassay (RIA) has many uses, including narcotics (drug)
detection, blood bank screening for the hepatitis a highly contagious
condition virus, early cancer detection, measurement of growth
hormone levels, tracking of the leukemia virus, diagnosis and
treatment of peptic ulcers, and research with brain chemicals called
neurotransmitters.
1) Detection of Narcotic Drugs- Heroin & Morphine can be detected in
hair with the use of Radioimmunoassay (RIA). In a research hair
samples obtained from morphine treated mice and heroin user
contained Nano gram levels of drug per milligram of hair . The result
of the hair analysis for all subject admitting the use of heroin were
positive where as the result of only 30% of thin layer chromatographic
urine analysis of these same subjects were positive.
2) Radioimmunoassay of Hydromorphone & Hydrocodone in Human
Plasma- Hydromorphone & Hydrocodone belongs to morphine group
of drugs and are used in combination with antitussive & analgesic
antipyretic mixture. The RIA method is capable of estimating the above
drug within a range of 2.5 to 20 ng/mL using standard 100 µl plasma
sample. RIA is carried out using morphine-6-antiserum &
dihydromorphine. Free drug is separated from the bound drug using
dextran coated charcoal & an aliquot of the supinate containing the
antiserum bound drug is subsequently counted for radioactivity.

3) Radioimmunoassay of Flunisolide in human plasma- Flunisolide is a
fast-acting corticoid designed for the treatment of allergic rhinitis,
asthma, and other allied respiratory disorders in humans. As the
quantum of drug delivered by inhalation (i.e., the usual route of
administration of the drug), is invariably small, the plasma-levels
attained can also be fairly small. Hence, there is a need for a sensitive
method of plasma concentration evaluation which is satisfied by
radioimmunoassay.
4) Measurement of Ferritin- Serum ferritin levels are indicative of iron
stores present in a patient. Levels are useful in differentiating true iron
deficiency from the body's failure to utilize these stores.
5) Detection of Digoxin- This allows direct measurement of serum
digoxin levels quickly and accurately. It is important to rule out Digi
toxicity quickly and accurately. We are also able to filter out Digi bind
to let the physician know how much the level has dropped after Digi
bind has been administered.
6) Thyroid Testing- This is used to determine the patient's thyroid status
and to follow patients after iodine-131 therapy to see if the dose was
indeed effective.

• Prolonged reaction time (in days) as a
consequence highly diluted reagent is used.
• Radioisotopes are costly.
• Possible health hazards due to handling of
radioisotopes.
• Limited assay range.
• Lack of direct linear relationship between
analyte concentration and signal response.
• Difficult of automation.
• Lengthy counting time.

1. Abraham, G.E. snd Grover, P.K. Covalent linkage of hormonal haptens
to protein carriers for use in radioimmunoassay. In Odell, W.D. and
Daughadsy, W.H. editors: Principles of competitive protein binding
assays. Philadelphia, J.B. Lippincott Co., 1971.
2. Rodbsrd, D., Rayford, P.L, Cooper, i.A. and Ross, G.T. Statistical
quality control and routine data processing for radioimmunoassays
(RIA) and immunoradiometric assays (IRMA). din. Chem.,
1974;20:1255-70.
3. Rodbsrd, D., Bridson, W. and Rayford, P.L Rapid calculation of
radioimmunosssay results. 3. Lab. dIm. Med., 1969;74:770-81.
4. Garcia, E.J. and Fiori, A. Radioimmunoasssy and competitive binding
analysis. In text book of nuclear medicine: BasicSciences (Eds. A.
Fernando snd.1.C. Harbert): Lea and Febiger Publishers, 1978; pp.
344360.
5. Hunter, W.M. and Greenwood, P.C. Preparation of 1-131 labeled
growth hormone of high specific activity. Nature (London),
1962;194:495-96.

ELISA and RIA

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