Introduction Principle, Requirements , Procedure and Application of Radioimmunoassay.

1. • Ankit Rajput
• Gmail- ar7310922@gmail.com
• Radioimmunoassay.
• Topic :- introduction, principle, requirements,
procedure, applications.
• Subject – Advanced Instrumentation Technique For
B.pharm 4 th year.

2. INTRODUCTION
• Radio immuno assay is a immnunological
assay to analyse antigens present in given
biological samples.
• It is most sensitive and specific method
of immuno assay.
• Senstivity ranges from 0.0006-0.006 ug/ml.
• It was developed by S.A. Berson and
Rosalyn yalow in 1959 in received
nobel prize in 1977

3. PRINCIPLES
It involves 3 principles which make it most
specific & sensitive than other immuno
assays.
An immuno reaction i.e. antigen, antibody
binding.
A competitive binding or
competitive displacement reaction.
Measurement of radio emission.

5. REQUIREMEN
TS
Micro titer plates / Test tubes
Pure antigen
Radio labelled of antigen
Antibodies
Standard's
Centrifuge
Radioactive counter

6. 1. MICRO TITER PLATE
Micro titer plate is commonly used
for this assay.
It could have 6, 24, 96, 384 or even
sometimes 1536 wells arranged in
rows.
Each well of a microtiter plate can
only hold very small amounts of
liquid.

7. PURE ANTIGENS
Antigens may be
obtained from
biological sample or
by synthetic form, it
should be pure.
It is used as standard
or calibrator.
RADIO LABELLING
OF ANTIGENS
The most commonly
used radiolabels are
tritium and iodine.
They have adequate
activity and have
long enough half
lifes.

8. ANTIBODY
1. Specific antibodies are obtained by
injecting Ag to animals.
2. Ag I.e. ,drug molecule + bovineserum albumin

9. CENTRIFUGE
1. Use for the separation of precipitated
form and supernatant liquid form.
2. Range :- 1200 – 2500 rpm.
RADIO ACTIVE COUNTERS
2 types of counters are used :-
• Gamma counters
• Scintillation counters

11. PROCEDURE
These test tubes
are incubated until
the reaction is
completed.
A competition
occur between Ag
& Ag* for the
binding sites of Ab.
This leads to
displacement
of Ag* by Ag.
Centrifugat
done for sepa
of bound an
form.

12. APPLICATIONS
• RIA of clonazepam
• RIA of barbiturates
• RIA of human plasma
• Determination of Ag concentration
• Estimation of hormones like LH, FSH, ACTH
• To detect hepatitis and HIV antigens
• Estimation of vitamins like folic acid , riboflavin
etc.

13. ADVANTAGES
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.

14. DISADVANTAGES
Radioactive iodine is used in is not a
cheap reagent.
Limited assay range.
Difficullty of automation.
Lengthy counting time.
All the reagents must be added
precisely.