Radioimmunoassay
Principle
Antigens and antibodies bind specifically to form the Ag-Ab complex. The antigen
can be labeled or conjugated with radioisotopes. The unlabeled antigens from the
sample compete with radiolabeled antigens to bind on paratopes of specific
antibodies. The unlabeled antigens replace labeled antigens that are already linked
with the antibodies. The unlabeled antigens when bind with antibodies, increases the
amount of free radiolabeled antigens in the solution. Hence the concentration of free
labeled antigens is directly proportional to the bound unlabeled antigens.
It involves a combination of three principles.
1. An immune reaction i.e. antigen, antibody binding.
2. A competitive binding or competitive displacement reaction. (It gives
specificity)
3. Measurement of radio emission. (It gives sensitivity)
Antigen- Antibody interaction
Steps involved in Radioimmunoassay.
The general steps involved in a radioimmunoassay are as follows:
• Preparation of reagents
• Formation of antibody-antigen complex
• Separation of bound and free fractions
• Measurement of radioactivity
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• Calculation of analyte concentration
Steps involved in radioimmunoassay.
1)Preparation of reagents:
The first step in RIA is the preparation of reagents. This includes labeling a known
quantity of the substance to be measured (the analyte) with a radioactive isotope,
typically iodine-125 (^125I) or tritium (^3H), to create the radiolabeled analyte. The
radiolabeled analyte is mixed with a specific antibody that is capable of binding to
the analyte.
2) Formation of antibody-antigen complex:
The radiolabeled analyte and the unlabeled analyte (in the sample to be measured)
compete for a limited number of antibody binding sites. When the sample containing
the analyte of interest is added to the mixture of radiolabeled analyte and antibody,
the analyte and radiolabeled analyte bind to the antibody, forming an antibody-
antigen complex.
3) Separation of bound and free fractions:
• The next step is to separate the bound and free fractions. This is typically done
using a separation method such as precipitation, filtration, or centrifugation.
• The bound fraction contains the antibody-antigen complex, while the free
fraction contains the unbound radiolabeled analyte and analyte from the
sample.
4) Measurement of radioactivity:
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• The radioactivity of either the bound or free fraction is measured using a
gamma counter, which is capable of detecting the radioactive emissions from
the radiolabeled analyte.
• The amount of radioactivity in each fraction is directly proportional to the
amount of antibody-antigen complex or unbound radiolabeled analyte,
respectively.
5) Calculation of analyte concentration:
• By comparing the radioactivity of the sample to that of a standard curve, which
is generated using known concentrations of the analyte, the concentration of
the analyte in the sample can be determined.
Applications of Radioimmunoassay
• Radioimmunoassay (RIA) is a powerful and versatile laboratory technique
used in various fields of science and medicine.
• Some of the applications of radioimmunoassay include:
1) Clinical diagnostics: RIA is widely used in clinical laboratories for measuring
the concentration of hormones, enzymes, drugs, and other small molecules in
patient samples. It is commonly used in the diagnosis and management of
endocrine disorders, such as thyroid diseases, diabetes, and hormonal
imbalances.
2) Pharmacokinetics and drug monitoring: RIA is used to study the
pharmacokinetics of drugs, which involves determining their absorption,
distribution, metabolism, and excretion in the body. It is also used for
therapeutic drug monitoring to optimize drug dosages and ensure safe and
effective treatment.
3) Cancer research: RIA is employed in cancer research for detecting and
measuring tumor markers, which are substances produced by tumor cells or
released into the blood circulation. Tumor markers can be used for cancer
diagnosis, prognosis, and monitoring response to therapy.
4) Veterinary medicine:
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RIA is used in veterinary medicine for measuring hormones, metabolites, and
drugs in animals. It is used in reproductive management, diagnosis of
hormonal disorders, and monitoring of drug therapy in animals.
5) Environmental science: RIA is used for monitoring environmental
contaminants, such as pesticides, heavy metals, and radioactive substances, in
soil, water, air, and biological samples. It is used in environmental research,
pollution control, and risk assessment.
6) Sports medicine and doping control: RIA is used in sports medicine for
detecting and measuring performance-enhancing drugs, such as anabolic
steroids, in athletes' urine or blood samples. It is also used in anti-doping
programs to ensure fair competition in sports.
7) Research in basic sciences: RIA is widely used in research laboratories for
studying various physiological and biochemical processes. It is used to
measure hormones, neurotransmitters, cytokines, and other molecules
involved in cellular signaling, metabolism, and immune response.
8) Agriculture and livestock management: RIA is used in agriculture and
livestock management for measuring hormones, nutrients, and drugs in plants
and animals. It is used for optimizing growth, reproduction, and health in crops
and livestock.
Questions
What is the principle of Radioimmunoassay (RIA)?
RIA is based on the competition between a radioactive-labeled substance (antigen or
antibody) and an unlabeled substance for binding sites on a specific antibody or
antigen.
How does RIA work?
• RIA involves the use of a radioactive substance, a known amount of antigen
or antibody, and a specific antibody or antigen to be measured.
• The radioactive and non-radioactive substances compete for binding sites on
the specific antibody or antigen
What is the role of radioisotopes in RIA?
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• Radioisotopes, such as iodine-125, are used to label either the antigen or
antibody.
• The radioactive signal allows for the detection and quantification of the
binding reactions.
What are the advantages of RIA?
RIA is highly sensitive, allowing for the detection of low concentrations of
substances. It is also specific, providing accurate measurements of particular antigens
or antibodies.
What are the applications of RIA?
RIA is widely used in clinical laboratories for measuring hormones, drugs, and other
biological substances. It is particularly valuable in endocrinology and oncology
How is RIA different from enzyme immunoassay (EIA)?
RIA uses radioactive isotopes for detection, while EIA uses enzymes. RIA tends to
be more sensitive, but EIA is safer and easier to handle due to the absence of
radioisotopes.
What are potential drawbacks of RIA?
The use of radioactive materials poses safety concerns, and proper handling and
disposal are essential. Additionally, RIA requires specialized equipment and trained
personnel.
How does RIA work?
Radioactively labeled and unlabeled substances compete for binding to a limited
number of antibody or antigen sites. The amount of radioactivity detected is inversely
proportional to the concentration of the substance being measured.
What is the main advantage of RIA?
RIA is highly sensitive, allowing the detection of very low concentrations of antigens
or antibodies in a sample.
Why might RIA still be used despite the availability of non-radioactive
alternatives?
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RIA is still used in specialized laboratories for its high sensitivity and specific
applications where the advantages outweigh the safety concerns associated with
radioactive materials.
Q: In RIA, what is labeled with a radioactive isotope?
Either the antigen or antibody is labeled with a radioactive isotope, such as Iodine-
125.
What is a potential drawback of RIA?
The use of radioactive isotopes in RIA raises safety concerns, requiring careful
handling and disposal procedures.
