SYNOPSIS INTRODUCTION PRINCIPLE HISTORY HOW TO RIA WORK METHOD APPLICATION OF RIA ADVANTAGE DISADVANTAGE CONCLUSION REFERENCES The technique in which a radioisotope is used as a tag or label (i.e. radioisotope covalently linked to antigen or antibody) for the detection of antigen-antibody complex is known as RIA. RIA involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantifitation using radioactivity. RIAs utilize a radioactive label (usually 125I, 3H or 14C), which emits radiation that can be measured with a beta or gamma counter.

1. RADIO IMMUNO ASSAY
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. SYNOPSIS
 INTRODUCTION
 PRINCIPLE
 HISTORY
 HOW TO RIA WORK
 METHOD
 APPLICATION OF RIA
 ADVANTAGE
 DISADVANTAGE
 CONCLUSION
 REFERENCES

3. INTRODUCTION
 The technique in which a radioisotope is used as a tag or label
(i.e. radioisotope covalently linked to antigen or antibody) for the
detection of antigen-antibody complex is known as RIA.
 RIA involves the separation of a protein (from a mixture) using
the specificity of antibody - antigen binding and quantifitation
using radioactivity.
 RIAs utilize a radioactive label (usually 125I, 3H or 14C), which
emits radiation that can be measured with a beta or gamma
counter.

4.  The technique of radioimmunoassay has
revolutionized research and clinical practice
in many areas, e.g.,
 ◦blood banking
 ◦diagnosis of allergies
 ◦endocrinology

5. RRINCIPLE
 RIA involves the competitive binding of a
radiolabelled antigen to a high affinity
antibody.
 The antigen is generally labelled with
Gama ray.

7. HISTORY
 The technique was
introduced in 1960 by
Berson and Yalow as
an assay for the
concentration of insulin
in plasma.
 It represented the
first time that hormone
levels in the blood could
be detected by an invitro
assay.

8. LABELS IN IMMUNOASSAYS- Immunoassays
require the use of labeled materials in order to
measure the amount of antigen or antibody present.
A label is a molecule that will react as part of the
assay, and in doing so produce a signal that can be
measured in the solution. Examples of a label include
a radioactive compound, or an enzyme that causes a
change of color in a solution or its fluorescence .
HOW RIA WORK

9. METHOD
 Classically, to perform a radioimmunoassay, a known quantity of
an antigen is made radioactive, frequently by labeling it with
gamma-radioactive isotopes of iodine, such as 125-I, attached
to tyrosine.
 This radiolabeled antigen is then mixed with a known amount
of antibody for that antigen, and as a result, the two specifically
bind to one another.
 Then, a sample of serum from a patient containing an unknown
quantity of that same antigen is added.
 This causes the unlabeled (or "cold") antigen from the serum to
compete with the radiolabeled antigen ("hot") for antibody
binding sites.
 As the concentration of "cold" antigen is increased, more of it
binds to the antibody, displacing the radiolabeled variant, and
reducing the ratio of antibody-bound radiolabeled

10. -Take a microtiter plate.
-Coated with antibody and specific to
the Ag.
-Washing to remove unbound Ag.

11. •After small amount of antigen added to the well
•Effect that competition b/w Ab and Ag.
•Removed hot Ab by cold Ag.
•After washing the unbound for unbound Ag
removed.
•Radioactivity is not 100%

12. •If 0 ng added to the well radioactivity is 100%
•When 1 ng unlabeled Ag will added it decreased
by 90%
•And the process is repeated all the process again
by increasing the
•It will be proportional to the concentration of
cold Ag.

13. •We can draw linear graph to the data correct
•And then to now determine concentration of A Ag present in sample.
•Take microtiter plate and coated with monoclonal Ab and again add
hot Ab.
•Instead we add sample there are Ag present in the sample

14. •According to the concentration unknown ag the will compete to the
radiolabel Ag, and remove them from the antibody.
•If radioactivity of the antibody is now 40%.we have to extrapolate
this value on standard graph.
•It will tell as concentration it 6 ng so our sample contain 6 ng of Ag.

15. The bound antigens are then
separated from the unbound ones,
and the radioactivity of the
free(unbound) antigen remaining
in the supernatant is measured
using a gamma counter.

16. APPLICATION OF RIA-
 RIA is used in the assay drugs like
digitoxin, morphin, barbifurate,
amphetonine.
 Analysis of vitamins like riboflavin, folic
acid.
 Analysis of hormone like aldosteron,
insulin, growth hormone, thyroxin.

17.  ADVANTAGE
 Radioimmunoassay is widely-used because of its great sensitivity.
 Using antibodies of high affinity, it is possible to detect a few
pictograms (10−12 g) of antigen in the tube.
 The greater the specificity of the antiserum, the greater the
specificity of the assay
 RIA has become a major tool in the clinical laboratory where it is
used to assay .
 Plasma levels of: ◦ most of our hormones; ◦ digitoxin or digoxin in
patients receiving these drugs; ◦ certain abused drugs.

18. DISADVANTAGE
 The main drawbacks to radioimmunoassay are the
expense and hazards if preparing and handling the
radioactive antigen.
 Both 125I or 131I emit gamma radiation that
requires special counting equipment;
 expensive instrumentation.

19. CONCLUSION
 Radio immune assay (RIA) is an elegant tech. in analytical
chemistry.
 If substance to be analysed is very low quantities, in the
order of microgram, conventional methods like gravimetric
and colorimetric method fail.
 RIA finds extensive application in the assay of many
substance which are present in trace amount in blood.

20. REFERENCES
 BOOK-
 Textbook of microbiology by Prescott, Harley.
 INTERNET SOURCE-
 http://www.antibodies-
online.com/resources/17/1215/radioimmunoassa
y-ria/
 http://www.biologynoteshelp.com/radio-
immuno-assaytypes-of-elisa/
 http://en.wikipedia.org/wiki/Radioimmunoassay
http://en.wikipedia.org/wiki/RIA
