This document provides an overview of radioimmunoassay (RIA), a sensitive and specific immunological technique for detecting antigens. It discusses the theory, principle, requirements, and procedure of RIA. RIA involves reacting an antigen with an antibody that has been radiolabeled. A competitive binding reaction then occurs between the radiolabeled and non-radiolabeled antigens. Measurement of radioemission allows for sensitive detection. RIA has applications in detecting hormones, drugs, toxins, and viruses in biological samples. Though sensitive, it requires specialized equipment and handling of radioactive materials poses health risks.

1. Sree Adichunchangiri college of Pharmacy
Immunological assays
Topic: Radioimmuno assay(RIA),
Presented by; Submitted to;
Prashanta Tavari Dr. Jaishree V
1st year M.pharm Prof,& Hoad
Department of Ph,ceutics Department of Pharmaceutical
SACCP Analysis
SACCP

2. RADIOIMMUNO ASSAY

3. Contents
 Introduction
 Theory
 Principle
 Requirements
 Procedure
 Application

4. Immunoassay
 An immunoassay is a test that uses antibody and
antigen complexes
 An antibody: Antigen complex is known as immune
complex
 Immune refers to an immune response that causes the
body to generate antibodies
 Immunoassay is a test that utilizes immuno
complexing when antibodies and antigens are brought
together.

5. Antibodies, Antigens.
An antibody is a protein that is produced by the body in response to an
“invading” (foreign) substance.
Antibodies are produced as a part of the body’s immue response to
protect itself.
An antigen is a toxin or other foreign substances which induces an
immune response in the body, especially the production of antibodies.
Ex, the drug is the antigen that binds to the antibody.
An immunogen is a substance that elicits immune response.
Ex, drug-protein conjugate

6. Introduction
 Radio immunoassay is an immunological assay to
analyses antigens present in given biological samples.
 It is most sensitive and specific method of
immunoassay
 Sensitivity ranges from 0.0006-0.006 µg/ml.
 It was development by S.A BERSON and ROSALYN
YALOW in 1959 and received nobel prize in 1977.

7. Theory
 RIA involves the reaction b/w antigen and its
specific antibody in presence of radio labelled
antigen.
 Animals normally develop Ab to the immunogenic
substances as a part of their natural immune
response.
 The serum derived from this animals is used as Ab
source and tested with reference to their
specificity, affinity at their titre level.

8. Principle
 This technique mainly based on the isotope dilution
principle, along with the use of specific anti body to
bind to a portion of the substance to be measure.
 It involves three principles which makes it most
specific and sensitive than other immune assays.
 A immune reaction i.e.,antigen, antibody, binding.
 A competitive binding or competitive displacement
reaction (it gives specificity).
 Measurement of radio emmission (it gives sensitivity)

10. Requirements
 Micro titer plate/ test tubes.
 Pure antigen
 Radio labeled antigen
 Antibodies
 Standards
 Centrifuge
 Radioactive counter

11. Micro titer plate
 A micro titer plate is
used mostly used for this
assay.
 Each well of of a
microplate can only hold
very small amounts of
liquid

12. Pure antigen
 Antigen may be obtained from biological sample like
serum, blood, harmones by synthetic form.
 It is used as standard or calibrator.
Radio labelling of antigen (Ag*)
The most common used radiolabels in RIA are tritium
(3H) and iodine(125 I )
They have adequate activity and have long enough half lifes.

13. Antibody
 The sensitivity and
sepcificity of RIA
depends on affinity Ag-
Ab reaction.
 In order to produce a
highly specific, the
antigen should have a
unique antigenic
determinant.

15. Centrifuge
 Used for the separation
of precipitated form and
supernatant liquid form
 Range 1200 – 2500 rpm

16. Radio active counters
 Two types of counters are used
 Gamma counter
 Scintillation counters

17. Gamma counter
 These are used for the gamma
energy emitting isotopes
 E.g., common iodine isotopes
Scintillation counter
They are used for counting beta
energy emitting isotopes
e.g., tritium, carbon-14 isotopes

19. Procedure
 These test tube are incubated until the reaction is
complete
 A competition occur b/w Ag and Ag* for the binding
sites of Ab.
 This lead to displacement of Ag* by Ag.
 Centrifugation is done for separation of bound and
free form.

20. Test tube- 1
BLANK
Ab+ Ag*
Test tube- 2
Calibrator
Ab + pure Ag + Ag*
Test tube- 3
Sample
Ab + sample Ag + Ag*
Procedure

21. Separation techniques
 After completion of reaction free form and bound
forms are determined by separation techniques.
 Various techniques include gel filtration,
electrophoresis, solid phase adsorption of Ag, Ab
and fractional precipitate.

22. Advantages
 Sensitive technique
 It can measure one trillionth of a gram of material per
milliliter of blood.
 Structurally specific as antigen: antibody reaction

23. Disadvantages
 Prolong time
 Radioactive iodine used is not cheap reagent
 Health hazards due to handling of radioisotopes
 Limited assay range
 All reagents should be precise

24. Applications
 Radio immuno assay of morphine includes
(a) synthesis of immunogen
(b) Antiserum production
(c) Procedure
 RIA of clonazepam, barbiturates, flurazepam in human
plasma
 RIA of hydrocarbon in human plasma
 Determination of Ag concentration
 Estimation of harmones like LH, FSH, ACTH
 To detect hepatitis and HIV antigens
 Estimation of vitamins like folic acid, riboflavin etc

25. Conclusion
 RIA is invitro assay technique used for the
determination of Ag concentration present in given
sample
 In advance to RIA many other techniques are
introduced like RAST (radio allergosorbent test), EIA
(enzyme immuno assay), competitive RIA

26. Reference;
 Skelley DS, Brown LP, Besch PK. Radioimmunoassay.
Clinical chemistry. 1973 Feb 1;19(2):146-86.
 Goldsmith SJ. Radioimmunoassay: Review of basic
principles. InSeminars in nuclear medicine 1975 Apr 1
(Vol. 5, No. 2, pp. 125-152). Elsevier.
 Images from www.google.co.in

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