2. Purpose is to determine the concentration of
an antigen in solution
Competitive binding assay
Originally developed by Yalow and Berson in
1960 for insulin
For her contribution of this important
analytical technique to medical science, R.
Yolow shared the 1977 Nobel price in
Medicine and physiology.
RIA
3. RIA
• Reagents
– Tracer: labeled antigen
– Antibody
– Standards: Known concentrations of unlabeled
antigen
– Unknown samples
6. •Separate bound from free:
•Antibody labeled tubes can be simply decanted
•Liquid-phase antibodies need to be precipitated
•Use a second antibody
•PEG
•Centrifugation
7. Count gamma emission
Counts per minute (CPM) for each tube
A sample containing a higher concentration of the
unknown antigen will have a lower CPM
8. Preparation of the Reagents:
Antibodies and Antigens
Polyclonal antibodies are made by injecting an animal with
the antigen, then purifying the antibody from serum.
Molecules smaller than ~1000 d are not generally
immunogenic
Steroids are covalently bond to protein carriers which
are immunogenic, antibodies can then be purified and
their specificity verified.
9. Preparation of the Reagents:
Iodination of the antigen
• I125 is the radioactive label most often used.
• Gamma emission at 35keV
• Available commercially as NaI
• Proteins with surface tyrosine groups can be oxidized with
commercially available products.
• I125 can be added to the tube and will bind to the oxidized
residues
• Column chromatography is used to purify the tracer
10. Normal range of the T4 concentration was determined in
the serum of men and women :
Detection limit:
It has been determined as being 2.5 ng/mL
(3.2 nmol/L).
An actual Assay by RIA-gnost® T4
11. An Actual Assay: T4 (thyroxine)
determination of the total thyroxine (T4) in human
serum by the principle of the competitive protein
binding analysis.
8-anilino-1-sulfonic acid (ANSA): T4 displacement from the
binding proteins and competes with I125 -T4 for binding
sites of a specific T4 antibody which are available in
limited numbers.
The quantity of bound T4 tracer is consequently inversely
proportional to the T4 concentration in the sample or
standard.
12. The test sample, i.e. the standard or the patient’s
serum are placed in the test tube (T4-antibody coated)
with the tracer and incubated.
13. Procedure
1. Mark the antibody-coated test tubes (duplicates for standards and
serum samples).
2. Pipette 20 μL standard or patient’s serum into the bottom of a test
tube.
3. In each case add 1 ml tracer.
4. The test tubes are shaken on a horizontal shaker for 2 hours (+/- 5
minutes) at room temperature.
5. Then remove the solution by decanting and place the tubes upside
down for 2 to 5 minutes on absorbent material. Remove remainders of
liquid adhering to the tube rim by briefly tapping. Alternatively, the
solution can be aspirated.
14. Measuring the radioactivity
The radioactivity adhering to the tubes is measured
for 0.5 – 1 minute in the I125 channel of a gamma
counter.
