This document describes detailed information about Radio immuno assay (RIA) including its principle, procedure, advantages, disadvantages, application etc

1. RADIOIMMUNOASSAY
PRESENTED BY- SK.AZIZ UDDIN
PRESENTED TO- DR.RIKESHWAR PRASAD DEWANGAN
M.PHARM,PHARAMACEUTICAL ANALYSIS
BATCH-2020-2022.
COLLEGE NAME-JAMIA HAMDARD

2. CONTENTS
 Introduction
 History of RIA
 Theory
 Principle
 Requirements
 Procedure
 Applications
 Advantages
 Disadvantages
 References

3. INTRODUCTION
 Radio immuno assay is an immunological assay
to analyse antigens present in given biological
samples.
 It is used as most sensitive and specific method
of immuno assay.
 Sensitivity ranges 0.0006-0.001 µg/ml.
 If substance to be analysed is in very low
quantities in the orders of
micrograms,nanograms, conventional methods
like gravimetric and colorimetric method fail but
RIA has extensive application in this
perspective.

4. HISTORY OF RIA
Developed by 1959 by
Rosalyn Yalow and
Solomon Berson for
measurement of Insulin
in plasma.
It represented the first
time that hormone levels
in the blood could be
detected by an in vitro
assay..
In 1977 Yalow received
the Nobel Prize for her
and Barson’s
development for RIA.

5. PRINCIPLE
 It is based on competitive binding between radio
labelled antigen(hot Ag) and unlabelled Ag (Cold
Ag) with selected antibody and ultimately radio
activity is determined.
 It basically involves three reaction i.e. antigen,
antibody binding.
 A competitive binding or competitive
displacement reaction(it gives specificity).
 Measurement of radio emission(it gives
sensitivity).

6. PRINCIPLE OF RIA
RIA is performed by using antibody-antigen binding and radioactive antigen. The
basic principle of RIA is competitive binding reaction, where the analyte(for
example antigen) competes with radio-labelled antigen for binding to the fixed
antibody or binding sites of receptor.

7. REQUIREMENTS
 Micro titre plate/ Test tubes.
 Pure antigen
 Radio labelled antigen
 Antibodies
 Standard’s
 Centrifuge
 Radioactive counter

8. MICRO TITRE PLATE
A microtitre plate is
mostly used for this
assay.
A microtitre plate
could have
60,24,96,384, or
even sometimes
1536 wells arranged
in rows.
Each well of a
microtitre can only
hold very small
amounts of liquid

9. ANTIGEN
 Pure Antigen
 Antigen may be obtained from biological sample
or by synthetic form. It should be pure.
 It is used as standard or calibrator.
 Radio labelling of antigen
 The most commonly used radio labelles in RIA
are tritium and iodine.
 They have adequate activity and have long
enough half lifes.

10. ANTIBODY
Specific antibodies are obtained by injecting the Ag to animals.
Ag I.e. Drug molecule + bovine serum albumin.

11. STANDARD
Draw a standard curve by taking conc. Of Ag in x- axis and radio
activity in y-axis.
It will help to determine the unknown conc. by using radio activity.

12. CENTRIFUGE
Uses for the separation of precipitated form and supernatant
liquid form.
Range: 1200-2500

13. RADIO ACTIVE COUNTERS
 There are two types of counters are used they
are:
1. Gamma counters-
 These are used for the gamma energy emitting
isotopes.
 E.g.- common iodine isotopes
1. Scintillation counters
 These are used for counting beta energy
emitting isotopes.
 E.g. – tritium, carbon 14 isotopes

14. GAMMA COUNTER VS SCINTILLATION COUNTER

15. PROCEDURE
 Step 1- In micro titre plate add antibody and add
radiolabelled Ag to it so all radiolabelled Ab will
bind to the Ag and forms Ag-Ab complex.
 Step 2- In this step add unlabelled Ag or cold Ag
and this cold Ag will replace the radio labelled
Ag and bind with Ab
 Step 3- Then wash with buffer solution and
washed solution will be collected in test tube
and do centrifugation process and supernatant
liquid will be analyzed for radio activity.

17. SEPARATION TECHNIQUE
 After completion of reaction 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.

18. APPLICATIONS
 Narcotics drug detection
 Early cancer detection
 Measurement of growth hormone levels.
 Tracking of leukemia virus.
 Diagnosis and treatment of peptic ulcers.
 Research with brain chemicals called
neurotransmitters.
 Estradiol measurement in translational studies
of breast cancer.

19. ADVANTAGES OF RIA
 Radio immuno assay is very sensitive
technique used to measure concentration of
Antigen without the need to use a bioassay.
 It is structurally specific as antigen; antibody
reaction are highly specific.
 It is indirect method of analysis.
 It is a saturation analysis as active reagent
added in smaller quantity than that of
analyte.

20. DISADVANTAGES OF RIA
 Radio active iodine used in is not a cheap
reagent.
 Possible health hazards due to handling of
radio isotopes.
 All the reagents must be added precisely.
 Limited assay range.
 Difficulty of automation
 Lengthy counting time.

21. REFERENCES
 Google slide share
 You tube- Dr. Pushpendra classes
 Immuno assay books.
Thank you