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RIA Notes

Radio Immuno Assay (RIA) is a sensitive technique developed by Berson and Yalow in 1960 for determining the concentration of antigens in biological fluids through antigen-antibody reactions and measuring radioactivity. The procedure involves using radiolabelled antigens and unlabelled antigens to create competition for binding sites on specific antibodies, allowing for quantification of antigens based on changes in radioactivity. RIA has applications in various fields including endocrinology and clinical immunology, but requires special expertise and is more expensive than other methods.

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228 views4 pages

RIA Notes

Radio Immuno Assay (RIA) is a sensitive technique developed by Berson and Yalow in 1960 for determining the concentration of antigens in biological fluids through antigen-antibody reactions and measuring radioactivity. The procedure involves using radiolabelled antigens and unlabelled antigens to create competition for binding sites on specific antibodies, allowing for quantification of antigens based on changes in radioactivity. RIA has applications in various fields including endocrinology and clinical immunology, but requires special expertise and is more expensive than other methods.

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prarthanasheth54
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Radio Immuno Assay (RIA)
Radio immuno assay can be used to determine the concentration of
antigens like harmones, serum protein, drug and vitamins in
biological fluids based on antigen antibody reaction and measuring
the radioactivity of resultant labelled antigen antibody complex.
This technique was developed by berson and yalow in 1960 they got
nobel prize in 1977
This technique is very sensitive that it can detect 0.001ug/ml
concentration of antigens

Principle:
The principle of RIA is based on antigen antibody reaction in which
the radio labelled antigen competes with the endogenous antigen for
the limited sites of the specific antibodies against the same antigen
the radio labelled antigen should have similar biological activity and
immunogenicity like that native antigen.

Antibody+ Antigen+ labelled antigen: Antigen- Antibody + labelled


antigen – Antibody + Antigen+ labelled antigen.

Procedure:
1. To carry out RIA procedure one require
•Anti A Antibodies: which bind to antigens in the sample which
are prepared by hybridoma technique
•Radiolabelled A Antigen: which some part is made up of Radio
isotopes so that they can emit raditation like alpha or beta,
heavy isotopes for H2
•Unlabelled antigens
2. Take a microtitre plate and coat with the antibodies
3. Then add Radio labelled antigens in excess amounts so that no
antigen bounding sites left free in which some Radio labelled
antigens are not bounded to the antibodies are removed by washing.
4. Hence the radioactivity of antigens –antibody is 100%
5. Then add known quantity of unlabelled antigen to the well which
creates the competition between labelled and unlabelled antigens
for binding with the antibody. This competition is proportional to the
concentration of unlabelled antigens added to the well.
6. Some labelled antigens will be removed from the antibody which
are replaced by unlabelled antigen and labelled and the radioactivity
of antibodies is not 100%
7. Repeat the procedure by increasing the concentration of
unlabelled antigens results in lesser radioactivity.
8. Plot a graph between concentration of unlabelled antigen and
Radioactivity of labelled antigen –antibody complex which gives a
linear graph.
9. Repeat the procedure by taking sample in place of unlabelled
antigen.
10. The antigens which are present in the sample will be compete
with the labelled antigen and forms a complex
11. The value of Radioactivity will give the concentration of antigens
present in the sample from the linear graph.

Applications of RIA:
1. RIA has been used to assay plasma levels of most hormones,
Insulin in human plasma, beta-HCG in females, vasopressin
2. Digitoxin or digoxin in patients receiving these drugs
3. Certain abused drugs like morphine
4. Presence of hepatitis B- surface antigen (HBS, Ag) in donated
blood, HIV.
5. Anti- DNA antibodies in systemic lupus erythematosus(SLE)

Merits of RIA:
1. High specificity and hence there is no interference from other
substances
2. High sensitivity
3. High precision
4. Applicable to wide variety of compounds in various fields
pharmacology, endocrinology, ancology, epidemiology and
clinical immunology
Demerits of RIA:

1. Special expertise and safety precautions are required, as radio


materials are used and disposed.
2. Expensive method compared to other methods as radio
materials are used.
3. Development of specific antibodies to the antigen

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