Radioimmunoassay (RIA) is an immunoassay technique that uses radiolabeled molecules to detect and quantify antigens or antibodies. RIA involves attaching a radioactive isotope to an antigen or antibody, then measuring the unbound antigen after the labeled and unlabeled antigens compete for binding sites on antibodies. RIAs are highly sensitive and can detect very low concentrations of substances like hormones, drugs, and vitamins in samples. They have various applications in disease diagnosis, drug monitoring, and pharmaceutical research.
2. Definition :
It is an immunological assay commonly used to measure antibodies, antigens,
proteins,harmones and glycoproteins in biological samples
ELISA is a distinguished analysis compared to other antibody-assays as it
yields quantitative results
3. ELISA works on the principle that specific antibodies bind the target
antigen and detect the presence and quantity of antigens binding. In order
to increase the sensitivity and precision of the assay, the plate must be
coated with antibodies/antigen with high affinity. ELISA can provide a
useful measurement of antigen-antibody concentration.
4. Generally the following apparatus is used for ELISA :
ELISA Kits
Pipettes
Pipette tips
Microplates
Spectrophotometer
Well wash microplate washer
5. ELISA assays are generally carried out in 96 well plates, allowing
multiple samples to be measured in a single experiment.
These plates need to be special absorbent plates (e.g. NUNC Immuno-
plates) to ensure the antibody or antigen sticks to the surface.
Each ELISA measures a specific antigen, and kits for a variety of
antigens are widely available.
6.
7.
8. • An antibody is attached to a polystyrene plate which is a solid surface
and is attracted or has an affinity towards bacteria, other antibodies and
hormones.
• A microtiter coated with antigen is filled with this antigen-antibody
mixture after which free antibodies are removed by washing.
• A second antibody specific to primary antibody is added which is
usually conjugated with an enzyme.
• Free enzyme-linked secondary antibodies are removed by washing the
plate.
• Finally, the substrate is added. The substrate is converted by the enzyme
to form a coloured product, which can be measured by
spectrophotometry.
9.
10. EXAMPLE:
HCG protein which indicates pregnancy is detected by ELISA. A
combination of blood or urine sample and purified HCG linked to an
enzyme is added to the system. If HCG is absent in the test sample, then
only the linked enzyme binds to the solid surface
11. ELISA tests can be classified into three types depending upon the different
methods used for binding between antigen and antibodies, namely:
• Indirect ELISA – Antigen is coated to the microtiter well
• Sandwich ELISA – Antibody is coated on the microtiter well
• Competitive ELISA – Microtiter well which is antigen-coated is filled
with the antigen-antibody mixture
12. Indirect ELISA :
• It detects the presence of antibody in sample. The antigen is attached to the
wells of the microtitre plate.
• A sample containing the antibodies is added to the antigen-coated wells for
binding with the antigen.
• The free primary antibodies are washed away and the antigen-antibody
complex is detected by adding a secondary antibody conjugated with an
enzyme that can bind with the primary antibody.
• All the free secondary antibodies are washed away. A specific substrate is
added which gives a coloured product.
• The absorbance of the coloured product is measured by spectrophotometry
13. Sandwich ELISA
• Sandwich ELISA helps to detect the presence of antigen in a sample.
• The microtitre well is coated by the antibody.
• The sample containing the antigen is added to the well and washed to
remove free antigens.
• Then an enzyme-linked secondary antibody, which binds to another
epitope on the antigen is added. The well is washed to remove any
free secondary antibodies.
• The enzyme-specific substrate is added to the plate to form a coloured
product, which can be measured
14.
15. Competitive ELISA
• Competitive ELISA helps to detect antigen concentration in a sample.
• The microtitre wells are coated with the antigen.
• Antibodies are incubated in a solution having the antigen.
• The solution of the antigen-antibody complex is added to the microtitre
wells. The well is then washed to remove any unbound antibodies.
• More the concentration of antigen in the sample, lesser the free antibodies
available to interact with the antigen, which is coated in the well.
• The enzyme-linked secondary antibody is added to detect the number of
primary antibodies present in the well.
• The concentration is then determined by spectrophotometry
16. Ebola
Pernicious anemia
AIDS
Rotavirus
Lyme disease
Syphilis
Toxoplasmosis
Zika virus
Carcinoma of the epithelial cells
17. The presence of antibodies and antigens in a sample can be determined.
It is used in the food industry to detect any food allergens present.
To determine the concentration of serum antibody in a virus test.
During a disease outbreak, to evaluate the spread of the disease
For Example, during recent COVID-19 outbreak, rapid testing kits are
being used to determine presence of antibodies in the blood sample.
19. INTRODUCTION
Radioimmunoassay is one of the sensitive immunoassay techniques
which helps in the determination of antigens or antibodies in a sample
with the use of radioisotopes.
A radioimmunoassay (RIA) is an immunoassay that uses radiolabeled
molecules in a stepwise formation of immune complexes.
RIA was first described in 1960 for the measurement of endogenous
plasma insulin by Solomon Berson and Rosalyn Yalow of the Veterans
Administration Hospital in New York.
20. It is an in vitro type of antigen-antibody interaction.
When radioisotopes instead of enzymes are used as labels to be conjugated
with antigens or antibodies, the technique of detection of the antigen-
antibody complex is called radioimmunoassay (RIA).
Radioimmunoassay (RIA) is an in vitro assay that measures the presence
of an antigen with very high sensitivity.
21. RIA is subdivided into four sections (primarily because there are four basic
steps in setting up an RIA procedure):
a) sample or standard
b) label or labeled antigen
c) primary binder
d) separation step.
22. Pipettors and/or pipets that accurately and precisely deliver the required
volumes
Polypropylene test tubes
Test tube rack
Vortex mixer
Beakers or flasks
Centrifuge (refrigerated, with swinging bucket rotor)
Liquid scintillation counter.
23. There are two different methods of RIA that are commonly employed for
drug detection.
Double-antibody RIA
Coated-tube RIA.
25. A radioimmunoassay (RIA) uses radiolabeled antigens to measure
concentrations of substances in body fluids such as blood and saliva.
A radioisotope is attached to an antigen of interest and bound with its
complementary antibody.
A sample with the target antigen is then added, which competes with the
radioactive antigen, kicks it out of the binding spot and replaces it.
26. After washing away unbound antigens the radioactivity of the sample is
measured. The smaller the radioactive signal, the more target antigen is
present. Compared to other immunoassay techniques, extra precautions are
needed due to the radioactive substances involved, but RIA’s high
sensitivity and specificity means that it remains in use today.
27. Fluoroimmunoassay
Fluorescent immunoassays (FIA) use a fluorescent compound as the
detection reagent to detect and quantify a variety of compounds.
FIA is widely used in the in vitro diagnostics (IVD) industry and has the
advantage of being fast and highly sensitive compared to other methods. In
FIA, antibodies are labeled with fluorescent probes.
FIA fluorescent dyes illuminate in UV light and are used to detect a
specific antigen-antibody binding. After incubation with the antigen, the
antibody-antigen complexes are isolated, and the fluorescent intensity is
measured.
28. Chemiluminescent immunoassay
is a variation of the standard enzyme immunoassay (EIA), which is a
biochemical technique used in immunology.
Applications
Chemiluminescent immunoassay (CLIA) has been applied in different
fields, including environmental monitoring, clinical diagnosis, food safety
and pharmaceutical analysis, as a promising approach for selective,
sensitive, rapid and simple analysis.
29. Counting immunoassay (CIA)
During incubation, the beads bind to a variety of antigens and
jointly form a large mass, but some beads are not bound. The
whole solution passes through a cell counter, with only
unbound beads counted. The amount of unbound beads is
inversely proportional to that of antigens.
30. GENERAL PROCEDURE OF RIA
A known quantity of an antigen is made radio active frequently by labeling
it with gamma radioactive isotopes of iodine attached to tyrosine. This
radio labeled antigen is then mixed with a known amount of antibody for
that antigen and as a result the two chemically bind to one another.
31.
32.
33.
34. CRP
CRP is an acute phase reactant and a general marker of systemic inflammation.
Well-known inflammatory markers includes,
C-reactive protein
Complete blood count
Albumin
Cytokines
Erythrocyte sedimentation rate.
36. The CD4+ T cell gene signature in progressors was enriched for
cytotoxicity-related genes GZMH, GZMB, GZMA, NKG7, FCRL6,
SLAMF7, CX3CR1, and PRF1, among others.
The most commonly mutated gene in people with cancer is p53 or
TP53. More than 50% of cancers involve a missing or damaged p53
gene.
Most p53 gene mutations are acquired. Germline p53 mutations
are rare, but patients who carry them are at a higher risk of
developing many different types of cancer.
37. Radioimmunotherapy (RIT) represents a selective internal radiation therapy, that
is, the use of radionuclides conjugated to tumor-directed monoclonal antibodies
(including those fragments) or peptides.
In a clinical field, two successful examples of this treatment protocol are
currently extended by 90Y-ibritumomab tiuxetan (Zevalin) and 131I-tositumomab
(Bexxar), both of which are anti-CD20 monoclonal antibodies coupled to
cytotoxic radioisotopes and are approved for the treatment of non-Hodgkin
lymphoma patients.
In order to reduce the unnecessary exposure and to enhance the therapeutic
efficacy, various biological, chemical, and treatment procedural improvements
have been investigated in RIT. This review outlines the fundamentals of RIT and
current knowledge of the preclinical/clinical trials for cancer treatment.
38. Radioimmunoassays are used for detecting the concentration of a specific
antigen.
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.
The advantages of RIA are its relative simplicity and the high sensitivity
provided by the use of radioactive compounds.
39. RIA is used in the assay of drug like
amphetamine,barbiturates,digitoxin,morphine etc. In analysis of vitamine like
riboflavin,folic acid.
Serum gastrin radioimmunoassay (RIA) is a sensitive and specific method
suitable for measurement of circulating concentrations of this peptide
hormone, which is a major regulator of gastric acid secretion.
The RIA technique may be useful for early (disease diagnosis) of viral
infections and for confirmation of response to immunization without the need
for a blood sample, as well as for the study of the secretory immune response
in very young and older subjects.
40. Radioimmunoassay (RIA) is a technique in which researchers use
radioactive isotopes as traceable tags to quantify specific biochemical
substances from blood samples.
Immunoassays have been widely used in many important areas of
pharmaceutical analysis such as diagnosis of diseases, therapeutic drug
monitoring, clinical pharmacokinetic and bioequivalence studies in drug
discovery and pharmaceutical industries.