RIA stands for Radioimmunoassay. It is an immunological assay.
It is basically used to determine the concentration of antigen in the blood serum of the patient with high sensitivity.
It has been utilized for quantitative assay of hormones, drugs, hepatitis B surface antigens, IgE and viral antigens, etc.
It has an application in the assay of substance which is present in trace amount in the blood.
The most common example of RIA is a RAST test (Radioallergosorbent test ). It is used to detect the causative antigen for allergy.
History of RIA (Radioimmunoassay)
RIA is developed by Rosalyn Yalow and Solomon Berson in 1959 for the measurement of insulin in plasma.
Since then it has been utilized to detect the hormone level in the blood by an in vitro assay. In 1977 Yalow was awarded by Nobel prize for her and Berson’s development of RIA.
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Principal of RIA
RIA is based on the competitive binding between the radiolabelled antigen ( hot antigen ) and unlabelled antigen (cold antigen)with a specific antibody.
The concentration of such labeled Molecules is determined by measuring their radioactivity rather than by chemical analysis.
Components used in RIA (Radioimmunoassay)
It is a labeled ligand. There are following isotopes are used in this assay as a tracer :
- Beta emitters – H ³ and C¹⁴
- Gamma emitters
3H – Tritium – It emits weak beta -ray. It requires low energy than C14 and if is produced by the neutron bombardment of lower hydrogen isotopes .it is used in the assay of drugs like proteins and amino acids .³H is very efficient for the usage of small samples because it has long shelf life (12.3yrs ) and high affinity. It has fewer health hazards. There are some limitations also i.e. it requires a scintillation counter which is costly and it has low specificity.
Carbon -14 – It also emits a weak beta ray. It is commercially available as barium carbonate.
Iodine -125 – It emits a low gamma-ray. It shows high specific activity. Iodine -125 is most favored in this assay because it found with high specific activity and almost 100% isotopic abundance thus reducing computing time and being economic. Its shelf life for labeled antigen is long. It can be easily introduced in the peptide molecules, steroids. Due to its gamma emission, it permits the simple less expensive instruments to determine the radioactivity.
It is a specific antiserum that is prepared by the injecting antigen intradermally into rabbits or Guinea pig, the production of antibodies takes place and then it recovered from serum.
Separation system – it is used because the bound antigen does not precipitate spontaneously at low concentrations. There are a variety of methods is used:
- Physical method – it includes centrifugation, electrophoresis, chromatography, etc
- Chemical method -some organic solvents are used in this essay such as ethanol, dioxane or salts such as zinc, sodium and ammonium sulfate.
Ligand free human serum – it can be obtained by treating human serum from charcoal and can also be obtained by collecting human serum from a volunteer in whom the production of ligand or hormones has been inhibited by the treatment with an appropriate drug.
The procedure used in RIA:
- Take a microtitre well which has attached antibody in it.
- Add radiolabeled antigen which is covalently bonded.(for example I-125)
- Incubate it.
- Allow the reaction to reach up to completion.
- Decant and wash the contents of the well by buffer such as 1% trifluoroacetic acid.
- Remove all the unbound antigens then only bounded form of antigen is available.
- Add the serum of the patient which contains unlabelled antigen.
- Then these unlabelled antigens will compete with labeled antigen in microtitre plate .and it replaces the labeled antigen by unlabelled antigen.
- Centrifuge the solution and use the supernatant for the analysis of radioactivity.
- Now measure the radioactivity remaining in the microtitre wells.
- The intensity of radioactivity is inversely proportional to the concentration of the antigens present in the test sample.
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Advantages of RIA (Radioimmunoassay)
- It is an extremely sensitive assay as it can measure antigen up to picogram quantities.
- It is a highly specific test as the antibody-antigen reaction is highly specific.
- A large number can be processed.
- It is an indirect method of analysis.
Disadvantages of RIA (Radioimmunoassay)
- Radiation hazardous .
- Require special arrangements for storage of radioactive material.
- The high cost of waste disposal.
- Lengthy counting time
- There are some difficulties in the automation of this assay.
- The reaction time is long due to the use of highly diluted reagent.
Instruments used in RIA
Centrifuge – There are two types of centrifuge used in RIA
- Swing bucket rotator –capable of generating 1200-2500 rpm.
- The pallet is formed at the bottom of the test tube.
- Fixed angle head rotor – capable of generating 3500-4000 rpm
- The pallet is formed at an angle
Radioactive counters – there are two types of radioactive counters used in RIA
- Gamma counters – used for counting gamma-energy isotopes, such as I-125.
- scintillation counters – used for counting Beta – energy isotopes .such as 3H, 14C.
Application of Radioimmunoassay (RIA)
- It is used in the assay of some drugs like morphine, digitoxin etc.
- It used to check the plasma level of hormones.
- Also used in the analysis of vitamins
- Also used in the analysis of anti- DNA antibody in systemic lupus erythematosus.
- It is also used in the early detection of cancer.
- Also used in the research of brain chemicals called neurotransmitter.
- And also used in the diagnosis and treatment of peptic ulcer.