Radioimmune Assay

1. SRI PARAMAKALYANI COLLEGE
( Reaccredited with A+ Grade with a CGPA of 3.9 in the III Cycle of NAAC
Affiliated to Manonmaniam Sundaranar University, Tirunelveli)
ALWARKURICHI -627 412
POST GRADUATE & RESEARCH CENTRE - DEPARTMENT OF MICROBIOLOGY
(Government Aided)
SUBMITTED TO
GUIDE: Dr. S.VISWANATHAN, Ph.D,
ASSISTANT PROFESSOR & HEAD OF THE DEPARTMENT,
SRI PARAMAKALYANI COLLEGE,
ALWARKURICHI.
K.Abinaya
Reg no 20221232516101
Ist msc microbiology
Sri paramakalyani college
Alwarkurichi

2. Radioimmunoassay is one of the sensitive immunoassay
techniques which helps in the determination of antigens or
antibodies in a sample with the use of radioisotopes.
It is an in vitro type of antigen antibody interaction.
When radioistopes instead of enzymes are used as labels to
be conjugated with antigens or antibodies, the technique of
detection of the antigen-antibody complex is called
radioimmunoassay.
Radioimmunoassay is an in vitro assay that measures the
presence of an antigen with very high sensitivity.
RADIO  Use of radio active material
IMMUNO  Antigen antibody binding theory
ASSAY  Detection of compounds

3. • Developed by in 1960 by two endocrinologists S.A. Berson and Rosalyn to determine
levels of insulin-anti insulin complexes in diabetics.
• It is used to measure
- Hormones
- Serum Proteins
- Drugs
- Vitamins at concentration of 0.001 micrograms/ml or less
• After the death of Berson the significance of the tech was acknowledge by the award of a
Nobel Price.
• The principle competitive binding of radiolableled antigen and unlabeled antigen to a high
affinity antibody.

4.  The principle of RIA involves competitive binding of
radiolabeled antigen and unlabeled antigen to a high affinity
antibody.
 The labeled antigen is mixed with antibody at a concentration
that saturates the antigen binding sites of the antibody.
 Then test samples of unlabeled antigen of unknown
concentration are added in progressively larger amounts.
 The antibody does not distinguish labeled from unlabeled
antigen, so the two kinds of antigen complete for available
binding sites on the antibody.
 As the concentration of unlabeled antigen increases, more
labeled antigen will be displaced from the binding sites.
 The decrease in the amount of radiolabeled antigen bound to
specific antibody in the presence of the test sample is
measured in order to determine the amount of antigen present

7.  The labelled antigen is mixed with the antibody that
saturates with antigen binding sites of the antibody. Then
test samples of unlableled antigen of unknown
concentration are added in progressively larger amounts.
 The antibody doesn’t distinguish labelled from unlabelled,
so there is competition between them for available
antigen binding sites on the antibody.
 As the concentration of unlabelled Ag increased more
labelled Ag are displaced from the binding sites.
 The decrease in the amount of radiolabeled Ag bound to
specific antibody in the presence of the test sample is
measured in order to determine the amount of Ag present
in the test sample.

8.  The Ag is generally labeled with gamma emitting isotope
such as 125I.
 But beta emitting isotopes such as tridtium are also
routineley used as labels.
 The radiolabled antigen is part of the assay mixture; The
test sample may be a complex mixture, such as serum or
other body fluids that contains the unlabeled antigen.
 The first step in the RIA is to determine the amount of Ab
needed to bind 50% to 70% of a fixed quantity of
radioactive ag in the assay mixture.
 This ration of Ab to labelled Ag is chosen to ensure that
the number of epitopes presented by the labeled Ag
always exceeds the total number of Ab binding sites.

9. • Consequently unlabeled Ag added to the sample mixture
will complete with radiolableled Ag for the limited supply
of Ab.
• Even a small amount of unlabeled Ag added to the
sample mixture to the assay mixture of a labeled Ag and
Ab will cause a decrease in the amount of radioactive Ag
bound, and this decreases will be proportional to the
amount of unlabelled Ag added.
• To determine the amount of labeled Ag bound, Ag-Ab
complexes is precipitated to separate it form free Ag and
the radioactivity in the precipitate is measured.
• A standard curve can be generated using unlabeled Ag
samples of know concentration and from this plot the
amount of Ag in the test mixture may be precisely
determined.

10. • Several methods have been developed for separating
bound Ag from in RIA.
• One method involves precipitating Ag-Ab complexes with
a secondary anti-isotype antiserum.
• For example, if the Ag-Ab complex contains rabbit IgG
Ab, then goat anti-rabbit IgG will bind to the rabbit IgG
and precipitate the complex.
• Another method makes use of the fact that protein A
Staphylococcus aureus has high affinity for IgG. If the
Ag-Ab complex contains an IgG antibody, the complex
can be precipitated by mixing with formalin-killed
S.aureus.

11. • After removal of the complex by either of these methods,
the amount of free labeled Ag remaining in the
supernatant can be measured in a radiation counter;
subtracting this value from the total amount of labeled
antigen added yields the amount of labeled Ag bound.
• Various solid-phase RIAs have been developed that
make it easier to separate the Ag-Ab complex from the
unbound Ag. In some cases, the Ab is covalently cross-
linked to beads.

12. • The amount of radio labeled Ag bound to the beads can
be measured after the beads have been centrifuged and
washed. Alternatively, the Ab can be immobilized on
polystyrene or polyvinyl chloride wells and the amount of
bound Ag determined.
• Because the procedure required only small amounts of
sample and can be conducted in small 96-well microtiter
plates, this procedure is well suited for determining the
concentration of a particular Ag in large numbers of
samples.

13. • For example, a microtiter RIA has been widely used to
screen for the presence of the hepatitis B virus.
• RIA screening of donor blood has sharply reduced the
incidence of hepatitis B infusions, in recipients of blood
transfusions.

14. 1. Radiolabeled antigens:
The antigens are generally labeled with gamma-ray
emitting isotopes such as I-125 and beta-ray emitting isotopes
such as Tritium. They are also called hot antigens.
2. Specific antibodies:
They are required in smaller amounts than antigens.
3. Unlabeled antigens (sample antigens):
They are also called cold antigens.
4. Microtiter plates:
96 wells microtiter plate
5. Washing Buffer solutions:
Wash buffer such as 1% Trifluoroacetic acid is used.

15. 1. Specific antibodies of known concentration are fixed in
the microtiter well.
2. A known amount of hot antigens is then added to the
well.
3. Washed carefully to remove any unbound antigens.
4. At this point, the radioactivity of the well will be
maximum.
5. Unlabeled antigens are then added to the well.
6. The unlabeled antigens will bind to the antibodies and
there will be free labeled antigens in the well.
7. Again washed carefully to remove the free labeled

17. It was first used for the detection of peptide hormones.
Detection of different viral antigens.
Detection of many hormones and drugs.
Detection of Hepatitis B surface antigens.
Detection of mycotoxins.
Detection of the early stage of cancer.

18. o High specificity
o High sensitivity
o Can detect a very small amount of antigen or antibodies.

19. o Difficulty of automation
o Lengthy counting time
o Limited assay range
o All the reagents must be added precisely
o Radioactive lodine used in is not a cheap reagent

20.  Working with radioactive substances makes it a bit risky.
 Disposal of radioactive substances can be problematic.
 Equipment and reagents are expensive.
 Radiolabeled substances used have a short shelf-life.

21. Refinement of Zalcitabine pharmaco kinetics.
Estradiol measurement in translational studies of breast
cancer.
Significance of prostate Specific antigen in prostate
cancer patients and in non cancerous prostatic disease
patients.
Evaluation of enzyme immunoassay and
radioimmunoassay methods for the measurement of
plasma oxytocin.

22.  Kuby immunology in 7th Edition.
Immunology Book.