The document discusses two types of immunoassays - ELISA and RIA. ELISA (enzyme-linked immunosorbent assay) is commonly used to detect antibodies, antigens, and proteins by immobilizing them on a microplate and detecting them using enzyme-linked antibodies. RIA (radioimmunoassay) uses radiolabeled antigens or antibodies to detect substances and can detect concentrations as low as nanograms or picograms per milliliter. Both techniques rely on the specificity of the antigen-antibody reaction and are used in clinical diagnostics, research, and quality control testing.
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.
This presentation explains about the Immunoassay ,radio immuno assay, definition, types, Principle , procedure, steps involved ,advantages ,disadvantages ,Application, RIA in insulin. RIA in Digitalis drug ligand etc....
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.
This presentation explains about the Immunoassay ,radio immuno assay, definition, types, Principle , procedure, steps involved ,advantages ,disadvantages ,Application, RIA in insulin. RIA in Digitalis drug ligand etc....
Enzyme immunoassays (EIAs), also known as enzyme-linked immunosorbent assays (ELISAs), combine antibody binding with enzymatic detection to quantify molecules of interest.
Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a 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. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal.
Spin-lattice & spin-spin relaxation, signal splitting & signal multiplicity concepts briefly explained relevant to Nuclear Magnetic Resonance Spectroscopy.
Flash Chromatography is a rapid form of preparative column chromatography based on an optimized pre-packed column through which is pumped solvent at a high flow rate.
The basic theory involved in the flash chromatography is similar to column chromatography except the involvement of pressure.
In column chromatography the flow of mobile phase is due to the gravitational force.
In flash chromatography the flow of mobile phase is under the influence of pressure.
Enzyme-Linked Immunosorbent Assay (ELISA) , Types of Elisa , Presentation on ...Rajesh Singh
ELISA stands for enzyme-linked immunosorbent assay.
It is a common laboratory technique which is used to measure the concentration of an analyte (antigens) in solution.
Where Ag-Ab interaction is monitored by enzyme measurement.
It is similar in principle to Radio Immuno Assay (RIA) but It depends on an enzyme rather than a radioactive label.
In this slide contains introduction, principle, types, equipment's and applications of Enzyme linked immunosorbent assay.
Presented by: D.Sudheer Reddy. (Department of pharmacology)
RIPER, anantapur.
ELISA use an enzyme to detect the binding of antigen (Ag) antibody (Ab). • The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab binding. • An ELISA can be used to detect either the presence of antigens or antibodies in a sample depending how the test is designed
ELISA, Principles of ELISA, Types of ELISA- Direct ELISA
Indirect ELISA, Sandwich ELISA, Competitive ELISA, and other Types i.e. ELISPOT (enzyme-linked immunospot assay) and In-cell ELISA, Advantages and disadvantages of ELISA detection methods, Different types of microplates for ELISA, Detection strategies for ELISA
Enzyme immunoassays (EIAs), also known as enzyme-linked immunosorbent assays (ELISAs), combine antibody binding with enzymatic detection to quantify molecules of interest.
Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a 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. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal.
Spin-lattice & spin-spin relaxation, signal splitting & signal multiplicity concepts briefly explained relevant to Nuclear Magnetic Resonance Spectroscopy.
Flash Chromatography is a rapid form of preparative column chromatography based on an optimized pre-packed column through which is pumped solvent at a high flow rate.
The basic theory involved in the flash chromatography is similar to column chromatography except the involvement of pressure.
In column chromatography the flow of mobile phase is due to the gravitational force.
In flash chromatography the flow of mobile phase is under the influence of pressure.
Enzyme-Linked Immunosorbent Assay (ELISA) , Types of Elisa , Presentation on ...Rajesh Singh
ELISA stands for enzyme-linked immunosorbent assay.
It is a common laboratory technique which is used to measure the concentration of an analyte (antigens) in solution.
Where Ag-Ab interaction is monitored by enzyme measurement.
It is similar in principle to Radio Immuno Assay (RIA) but It depends on an enzyme rather than a radioactive label.
In this slide contains introduction, principle, types, equipment's and applications of Enzyme linked immunosorbent assay.
Presented by: D.Sudheer Reddy. (Department of pharmacology)
RIPER, anantapur.
ELISA use an enzyme to detect the binding of antigen (Ag) antibody (Ab). • The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab binding. • An ELISA can be used to detect either the presence of antigens or antibodies in a sample depending how the test is designed
ELISA, Principles of ELISA, Types of ELISA- Direct ELISA
Indirect ELISA, Sandwich ELISA, Competitive ELISA, and other Types i.e. ELISPOT (enzyme-linked immunospot assay) and In-cell ELISA, Advantages and disadvantages of ELISA detection methods, Different types of microplates for ELISA, Detection strategies for ELISA
This presentation explains about the principle and procedure involved in elisa method of immunoassay, development o f elisa , application advantages and disadvantages of elisa
A path breaking technology which has made it possible for us to detect HIV. ELISA is an immunological assay nowadays even used to detect food proteins & is the science behind pregnancy color test. This presentation unlocks the working of this assay.
ELISA, principle and method by kk sahuKAUSHAL SAHU
What is ELISA.
Principle.
History.
Types of ELISA method.
1.Direct ELISA.
2.Indirect ELISA.
3.Sandwhich ELISA.
Conclusion.
References.
Antibodies (also known as immunoglobulins abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Comparative structure of adrenal gland in vertebrates
Elisa ria
1. KUVEMPU UNIVERSITY
Sahyadri Science College, Shimoga
PG Department of Biotechnology
Topic : Immunoassays - ELISA and RIA
PRESENTED BY UNDER THE GUIDANCE
Ms. Bi Bi Ayesha Dr. Jamuna K S
I MSc, II Sem Lecturer
Sahyadri Science College Sahyadri Science College
Shimoga Shimoga
2. Contents
Introduction
ELISA
Introduction
History
Principle of ELISA
Procedure of ELISA
Types of ELISA
Advantages of ELISA
Disadvantages of ELISA
Applications of ELISA
RIA
Introduction
History
Principle of RIA
Procedure of RIA
Advantages of RIA
Disadvantages of RIA
Applications of RIA
Conclusion
References
3. Introduction
Immunoassays are bioanalytical methods that use the specificity of an antigen-
antibody reaction to detect and quantify target molecules in biological samples.
These methods are frequently used in clinical diagnostics, drug discovery, drug
monitoring, and food testing.
The first immunoassay developed was described by Yalow and Berson.
They used radiolabelled insulin to assess the concentration of insulin in human
plasma, and thus developed the first radioimmunoassay (RIA).
Antigens were immobilized on a microplate well, incubated with antiserum, and
then the concentration of antibody in the antiserum was quantified using an
enzyme-linked anti-immunoglobulin antibody. This method is the enzyme-linked
immunosorbent assay (ELISA).
5. Enzyme-linked immunosorbent assay (ELISA)
ELISA is an immunological assay commonly used to measure
antibodies, antigens, proteins and glycoproteins in biological
samples.
Originally described by Peter Permann and Eva Engvall (1971),
the method enables analysis of protein samples immobilized in
microplate wells using specific antibodies.
ELISAs are typically performed in 96-well or 384-well
polystyrene plates, which passively bind antibodies and
proteins.
ELISA has been used as a diagnostic tool in medicine, plant
pathology, and biotechnology, as well as a quality control check
in various industries.
7. Principle of ELISA
The most crucial element of an ELISA is a highly specific
antibody-antigen interaction.
Antigen of interest isabsorbed on to surfacesorbent
Antigenisrecognized onlybythespecific antibodyimmune
Thisantibody isrecognized by second antibody (immuno)which has
enzyme attached (enzyme linked)
Hencethename,Enzyme-linked immunosorbent assay
Substrate reacts with enzyme to produce product usually
depicted by coloured.
The development of colour using a chromogenic substrate
corresponds to the presence of the antigen.
8. In an ELISA, an enzyme is linked to an antibody that has been
specifically raised against an antigen of interest.
Biological samples containing the target antigen are allowed
to bind the enzyme-labeled antibody and the reaction is
visualized using an enzyme-specific substrate which is added
to the reaction mixture.
The enzyme-specific substrate binds to the enzyme attached
to the antibody, generating a colored product which can be
detected using chromogenic or chemiluminescent imaging
techniques.
ELISA has a high specificity and sensitivity, thus it is frequently
used for high-throughput screening of antibodies or drugs.
Principle of ELISA
9. The most commonly used enzyme labels are horseradish peroxidase (HRP)
and alkaline phosphatase (AP).
Other enzymes have been used as well; these include β-
galactosidase, acetylcholinesterase, and catalase.
A large selection of substrates is available commercially for performing ELISA
with an HRP or AP conjugate.
The choice of substrate depends upon the required assay sensitivity and the
instrumentation available for signal-detection (spectrophotometer,
fluorometer, or luminometer).
10. Procedure of ELISA
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.
11. Types of ELISA
ELISA tests can be classified into three types depending upon the different
methods used for binding between antigen and antibodies, namely:
Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA
12. Direct ELISA
In a direct ELISA, the antigen is bound to the bottom of the microplate well, and then
it is bound by an antibody that is specific to the antigen and also conjugated to an
enzyme or other molecule that enables detection.
13. Indirect ELISA
In an indirect ELISA, the antigen is bound to the bottom of the microplate
well, then an antibody specific to the antigen is added.
A secondary antibody, conjugated to an enzyme or other detection molecule,
is then bound to the first antibody.
14. Sandwich ELISA
For the sandwich ELISA, two antibodies specific to two different epitopes on the
target antigen are used.
The capture antibody is bound to the bottom of the microplate well and binds one
epitope of the antigen.
The detection antibody binds to the antigen at a different epitope and is conjugated
to an enzyme that enables detection. (If the detection antibody is unconjugated,
then a secondary enzyme-conjugated detection antibody is required).
15. Competitive ELISA
In a competitive ELISA, a reference antigen is bound to the bottom of microplate wells.
Sample plus antibody are added to the wells, and if there is antigen present in the
sample, it competes with reference antigen for binding to the antibody. Unbound
material is washed away.
The more antigen was in the sample, the less antibody ends up bound to the bottom
of the wells by the reference antigen, and the lower the signal.
17. ELISPOT
(enzyme-linked immunospot assay)
It refers to ELISA-like capture and measurement of proteins secreted by cells
that are plated in PVDF-membrane-backed microplate wells.
It is a "sandwich" assay in which the proteins are captured locally as they are
secreted by the plated cells, and detection is with a precipitating substrate.
ELISPOT is like a western blot in that the result is spots on a membrane
surface.
18. In-cell ELISA
In-cell ELISA is performed with cells that are plated and cultured overnight in
standard microplates.
After the cultured cells are fixed, permeabilized, and blocked, target proteins
are detected with antibodies.
This is an indirect assay, not a sandwich assay.
The secondary antibodies are either fluorescent (for direct measurement by a
fluorescent plate reader or microscope) or enzyme-conjugated (for detection
with a soluble substrate using a plate reader).
19. Variants of ELISA
Although many variants of ELISA have been developed and used in different
situations, they all depend on the same basic elements:
Coating/capture – direct or indirect immobilization of antigens to the
surface of polystyrene microplate wells.
Plate blocking – addition of irrelevant protein or other molecule to
cover all unsaturated surface-binding sites of the microplate wells.
Probing/detection – incubation with antigen-specific antibodies that
affinity-bind to the antigens.
Signal measurement – detection of the signal generated via the direct
or secondary tag on the specific antibody.
20. ELISA plates
ELISA is nearly always performed using 96-well or 384-well polystyrene plates
and samples in solution (i.e., biological fluids, culture media, or cell lysates).
When developing a new ELISA for a specific antigen, the first step is to
optimize the plate-coating conditions for the antigen or capture antibody.
Plate coating is achieved through passive adsorption of the protein to the
plastic of the assay microplate. This process occurs though hydrophobic
interactions between the plastic and non-polar protein residues.
21. Different types of pre-coated microplates for
ELISA
ELISA plate Coating Applications
Modified polymer surfaces Various modifications to the plate
surface to increase hydrophobicity or
hydrophilicity
Enhance passive binding of biomolecules
based on their physiochemical characteristics
Antibody-binding plates Protein A, G, L, or A/G Binds to the FC region (VL for protein L) of
capture antibodies to properly orient while
leaving antigen binding capability
Biotin-binding plates Streptavidin or neutravidin Binds small biotinylated peptides and other
small molecules that are difficult to bind by
passive adsorption
Fusion-tag binding plates Glutathione (GST tag binding) or nickel
or copper coated (His tag binding)
Study of genetically engineered fusion
proteins or protein-protein interactions
22. Detection strategies for ELISA
Chromogenic
(colorimetric)
Fluorescence Chemiluminescence
Sensitivity
Equipment required Standard absorbance plate
reader
Fluorometer Luminometer plate reader
Enzyme HRP or AP Fluorescent tag or HRP
(with chemifluorescent
substrates)
HRP or AP
Advantages Direct visualization, high
reproducibility between
plates
High reproducibility
between plates, wide
dynamic range
Most sensitive detection
strategy, wide dynamic
range
Considerations Requires black microplates Requires opaque or black
microplates
23. Advantages of ELISA
Results fetched from ELISA gives an accurate diagnosis of a particular disease
since two antibodies are used.
Can be carried out for complex samples as the antigen is not required to get
purified to detect.
It is highly responsive since direct and indirect analysis methods can be
carried out.
It is a rapid test, yields results quickly.
Possible detection for ELISA ranges from the quantitative, semi-quantitative,
standard curve, qualitative, calibration curve models etc.
Easier to perform and uncomplicated process as compared to other assays
which require the presence of radioactive materials.
24. Disadvantages of ELISA
Labour – intensive and expensive to prepare antibody
Sophisticated techniques and expensive culture media are required.
High possibility of false positive/negative.
Insufficient blocking of immobilized antigen results in false results.
Antibody instability.
Refrigerated transport and storage are required as an antibody is a
protein.
25. Applications
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.
Several diseases are diagnosed using ELISA - e.g. Ebola, Pernicious anaemia, AIDS,
Rotavirus, Lyme disease, Syphilis, Toxoplasmosis, Zika virus, Carcinoma of the epithelial
cells, SARS-CoV-2 etc.
During a disease outbreak, to evaluate the spread of the disease, e.g. during recent
COVID-19 outbreak, rapid testing kits are being used to determine presence of antibodies
in the blood sample.
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.
The more the substance of interest is present, the more reaction takes place and less of
linked enzyme binds to the solid surface. These reactions are indicated usually with a
change in the colour of the solution.
27. Radioimmunoassay (RIA)
A radioimmunoassay (RIA) is an immunoassay that
uses radiolabeled molecules to quantify hormones, drugs, and viral antigens.
A RIA is a very sensitive in vitro assay technique used to measure
concentrations of substances, usually measuring antigen concentrations by
use of antibodies.
The radioimmunoassay technique, as the name implies, achieves sensitivity
through the use of radionuclides and specificity that is uniquely associated
with immunochemical reaction.
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.
28. History
Developed in 1959 by Rosalyn Yalow and
Solomon Berson for measurement of
insulin in plasma.
It represented the first time that hormone
levels in the blood could be detected by an
in vitro assay.
In 1977 Yalow received the Nobel Prize for
her and Berson’s development of RIA
29. Radioimmunoassay can detect
substance like :
• Hormones
• Vitamins
• Serum Protein
• Drugs
• Infective Agent
Sensitivity of
Radioimmunoassay :
It can detect substance from a
range of Nano gram(ng) to Pico
gram(pg) concentration.
Qualitative as well as Quantitative analysis.
30. Principle of Radioimmunoassay (RIA)
Radioimmunoassay uses radioisotope-labeled purified known antigen which
competes with unlabeled (unknown) antigen for binding sites on a known
amount of antibody.
The Ag-Ab complexes that form between the antigen and antibody can then be
precipitated using the second antibody and the amount of radioactivity of the
bound complex is measured by means of radioisotope analyzers and
autoradiography.
31. Radioimmunoassay (RIA) method
Radioimmunoassay is an old assay technique but it is still a
widely used assay and continues to offer distinct advantages
in terms of simplicity and sensitivity.
RIA is a competitive binding assay. The target antigen is
labelled radioactivity and bound to its specific antibodies.
Radioactivity: 125-I labels are usually applied although other
isotopes such as C14 and H3 have also been used.
The antibody & labelled antigen are always present as
limiting factors and the concentrations of unlabelled antigens
(sample) under examination is increased continually
32. Advantages of RIA
Radio immune assay is very sensitive technique used to measure
concentration of antigen without the need to use a bioassay.it can measure
one trillionth (10-12) of a gram of material per millilitre of blood.
It is structurally specific as antigen: antibody reaction are highly specific.
It is indirect method of analysis
It is a saturation analysis as active reagent added in smaller quantity than that
of analyte.
High possible to detect pictogram of antigen
33. Disadvantages of RIA
Prolonged reaction time (in days) as a consequence highly diluted reagent is used.
Radioisotopes are costly.
Possible health hazards due to handling of radioisotopes.
All the reagents must be added precisely.
Limited assay range.
Lack of direct linear relationship between analyte concentration and signal response.
Difficulty of automation.
Lengthy counting time.
34. Applications of RIA
Endocrinology
Insulin, HCG, Vasopressin
Detects endocrine disorders
Physiology of endocrine function
Pharmacology
Morphine
Detect drug abuse or drug poisoning
Study drug kinetics
Epidemiology
Hepatitis B
Clinical Immunology
Antibodies for inhalant allergens
Allergy diagnosis
Oncology
Carcinoembryonic antigen
Early cancer detection and diagnosis
