1. RADIOIMMUNOASSAY (RIA)
Dr. Sigal Fleisher-Berkovich
Dept. of Clinical Pharmacology

2. Principles of the Radioimmunoassay
A fixed concentration of labeled tracer antigen is incubated with a constant amount of antiserum
If unlabeled antigen is added to the system, there is competition between labeled tracer and unlabeled antigen for the limited number of binding sites on the antibody
The amount of tracer bound to antibody will decrease as the conc. of unlabeled antigen increases
This can be measured after separating antibody bound from free tracer and counting the bound fraction

3. RIA
radioactivity
Increasing amount of Insulin

4. Four basic necessities for RIA
Antiserum to the compound to be measured
2. A radioactively labeled form of the compound
3. A method whereby antibody-bound tracer can
be separated from “free” tracer
4. A standard unlabeled material

5. Terminology Total Count Tubes:
These tubes represent the total amount of radioactivity added in a RIA tube.
They have the highest CPM’s (labeled antigen +buffer)
Role: quality control to the counts in the rest of the tubes

6. Terminology Background (blank) tubes:
These tubes contain buffer, labeled antigen
and charcoal but not antibody
They have the lowest CPM’s
These counts are considered to be background counts

7. Terminology Standards: Assay tubes that contain known amounts of
the compound to be measured.
Those tubes with increasing amount of standard have decreasing CPM’s.
RIA curve

8. Terminology Unknowns:
Assay tubes containing the material in which the compound you wish to measure is contained.
Blood serum & plasma
Urine
Saliva
CSF

9. Terminology Precipitating agent: It is added at the end of incubation
It precipitates the “free” labeled and unlabeled antigen
Our precipitating agent is dextran-coated charcoal

10. RIA Table No of tubes: 2 2 4 2 2 buffer (ml) 0.8 0.6 0.1 - -
Total Blank Zero standard assay
No of tubes:
buffer (ml)
Standard (ml)
Sample (ml)
Antibody (ml)
Incubation for 30 min
Labeled PG (ml)
Incubation for 1-24 hr
Charcoal

11. RIA Advantages: Extremely sensitive method
Large number of samples can be processed
Small changes in hormone concentrations can be
reproducibly measured
Disadvantages:
Can’t determine if hormone measured has biological
Activity
Peptide hormones can be denatured and not active but still
retain their antigenic character

12. Prostaglandin E2
Prostaglandins: a 20-carbon unsaturated fatty acids derived from arachidonic acid
The inflammatory response is always accompanied by the release of prostaglandins, the predominant product being PGE2
PGE2 mediates: fever, pain
vasodilation