radioactive techniques

1. RADIOACTIVE
TECHNIQUES
PRESENTED BY
T.HEMANTH
There is no elevator to success, You have to take
the stairs.

2. Overview:
• Basic concepts
• Radioimmunoassay- RIA
• Immunoradiometric Assay- IRMA
• Radioallergosorbent Test- RAST
• Toxicity and safety aspects

3. RADIOACTIVE
• RADIOAVTIVE is the spontaneous emission of
radiation in the form of particles or high energy
photons resulting from nucler reaction.
• A substance that contains unstable atomic
nuclei is considered to be radioactive.
Paricles/ Radiation
Unstable Stable

4. RADIOACTIVE DECAY
• Radioactive decay is the process by which an
unstable atomic nucleus loses energy by radiation.
TYPES OF RADIOACTIVE DECAY:
• Alpha decay : occurs when the nucleus ejects an
alpha particle ( helium nucleus)
• isotopes of elements with high atomic numbers
• High energy and ionisation but least penetration
power.

5. • Cancer Treatment: source radiotherapy (radium-226)
• Static eliminator: eliminate static electricity in
industrial application.
• Smoke detector: smoke particles disrupt this current,
triggering an alarm.
• Pacemaker battery : used as an energy source to
power for heart pacemakers (Plutonium-238 hf life 88
yr)
• Spacecraft Power (Plutonium-238)
• Remote Sensing Stations (Strontium-90)

6. Beta decay : two ways
a) β-minus decay : neutron to proton
b) β- plus decay : proton to neutron
• used in science and medicine particularly in the
field of oncology, PET imaging and cancer
diagnosis.
Gamma Decay :
• Electromagnetic radiation & extremely short
wavelength
• Low ionising power, high penetration
• Accompanies other particles emissions.

7. POSITRON EMISSION:
• It is subtype of radioactive decay called beta decay.
Contain positively charged beta particles.
• Positron emission tomography (PET) scan is an imaging
test that helps detect organs and tissues function.
ELECTRON CAPTURE :
• Electron in nucleus combines with proton forming a
neutron and a neutrino.
• Electron capture detectors (ECD) used in environmental
testing.

8. Radioactive techniques in clinical
chemistry
Those are
• Radioimmuno assay (RIA)
• Immunoradiometric assay (IRMA)
• Radioallergosorbent test (RAST)
• Isotope dilution mass spectrometry
(IDMS)

9. RADIOIMMUNOASSAY (RIA)
• Radioimmunoassay (RIA) is a very sensitive in vitro
assay technique used to measure concentrations of
antigens (for example, hormone levels in the blood)
by use of radioactive antibodies.
• RIA technique is extremely sensitive and specific,
requiring specialized equipment
• The technique was introduced in 1960 by berson
and yalow as an assay for the concentration of
insulin in plasma

10. PRICIPLE
The technique is based on the ability of an unlabeled
form of the substance to inhibit competitively the binding
of a radioactively labelled substance by specific
antibodies.

11. REQUIREMENTS:
1. Pure antigen : for - standards (μg).
2. Tracer : self-made or commercial.
3. specific, high-affinity antibody : selfmade or
commercial.
4. A method to separate bound and free antigen.
5. (Optional) : A system to extract the antigen from
the sample.

12. METHOD
• To perform a radioimmunoassay, a known
quantity of an antigen is made radioactive,
frequently by labeling it with gamma-radioactive
isotopes of iodine attached to tyrosine (hot).
• This radiolabeled antigen is then mixed with a
known amount of antibody for that antigen.
• Then, a sample of serum from a patient
containing an unknown quantity of that same
antigen is added.

13. • 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 antigen to free radiolabeled
antigen.

14. • The bound antigens are then separated from the
unbound ones, and the radioactivity of the free
antigen remaining in the supernatant is
measured using a gamma counter.
• a binding curve can then be generated which
allows the amount of antigen in the patient's
serum to be derived

15. Separating Bound from Free Antigen
Precipitate the antigen-antibody complexes by adding a
"second" antibody directed against the first. For
example, if a rabbit igg is used to bind the antigen, the
complex can be precipitated by adding an antirabbit igg
antiserum.

16. Radioimmunoassay: pros and cons
PRO:
• versatility : using the same principle, almost any
biomolecule can be assayed
• Fast (usually 2 days or less)
• Sensitive (comparable to the most sensitive
bioassays, that is < ng/ml)
• Large capacity : thousands of samples/day specific
(antibodydependent)

17. Con :
• Use of radioactivity: hazardous
• Expensive equipment (gamma or beta Counter)

18. IMMUNORADIOMETRIC ASSAY
• INTRODUCTION
Fluorescent and radioactive antibodies have been
used to measure solid-phase antigens.

19. PRINCIPLE
• In IRMA, the antibodies are labeled with radioisotopes
which are used to bind antigens present in the
specimen.
• When a positive sample is added to the tubes,
radioactively antibodies bind to the free epitopes of
antigens and form an antigen-antibody complex.
• Unbound labeled antibodies are removed by a second
reaction with a solid phase antigen. The amount of
radioactive remaining in the solution is directly
propotional to the antigen concentration in the sample.

21. RADIOALLERGOSORBENT TEST (RAST)
The RAST is a radioimmunoassy test to detect IgE
antibodies to suspected or known allergens for the
purpose of guiding a diagnosis about allergy.
Method
• The suspected allergen is bound to an insoluble
material and the patient serum is added.
• If the serum contains antibodies to the allergen
those antibodies will bind to the allergen.

22. • Radiolabeled anti- human IgE antibody is added
where it binds to those IgE antibodies alredy
bound to the insoluble material .
• The unbound anti-human IgE antibodies are
washed away.
• The amount of radioactivity is proportional to the
serum IgE for the allergen.

23. RAST rating IgE level (kU/L) comment
0 < 0.35
ABSENT OR
UNDETECTABLE
ALLERGEN SPECIFIC IgE
1 0.35 – 0.69
LOW LEVEL OF ALLERGEN
SPECIFIC IgE
2 0.70 – 3.49
MODERATE LEVEL OF
ALLERGEN SPECIFIC IgE
3 3.50 – 17.49
HIGH LEVEL OF
ALLERGEN SPECIFIC IgE
4 17.50 – 49.99
VERY HIGH LEVEL OF
ALLERGEN SPECIFIC IgE
5 50.00 – 100.00
ULTRA HIGH LEVEL OF
ALLERGEN SPECIFIC IgE
6 > 100.00
EXTREMELY HIGH LEVEL
OF ALLERGEN SPECIFIC
IgE

24. ISOTOPE DILUTION MASS SPECTROMETRY
• It is a reference technique for quantitative analysis.
• the precision and accuracy associated with the use of
internal standards.
• A major complication of isotope dilution mass
spectrometry proteomics is the technical difficulty of
obtaining these internal standards

25. SAFETY

26. Summery
• RADIOAVTIVE is the spontaneous emission of radiation
in the form of particles.
• RADIOACTIVE DECAY is mainly 3 types alpha, beta and
gamma.
• Radioactive techniques used in clinical chemistry
are radioimmunoassay, immunoradiometric
assay, radioallergosorbent assay, isotope dilution
mass spectrometry.