What is RIA, Principle of RIA, Techniques used, Pros and Cons of RIA, RIA Applications

1. Radioimmunoassay (RIA)
and its Application in Detection Of
Prostatic Cancer
Teacher-In Charge
Mr. Tushar Khare
- Akshay More

2. Outline
 Introduction
 Principle
 Technique
 Pros and Cons
 Application

3. Introduction
 The most sensitive techniques for detecting antigen.
 First developed by two endocrinologists, S. A. Berson and Rosalyn Yalow (1960)
for determination of insulin–anti-insulin complexes in diabetics.
 Definition:
“A Binding Assay....
in which the binder is an antibody which uses radioactivity to measure the amount
of bound and/or free antigen”
 Radioactively labelled antigen is called "tracer"
 Radioactive isotopes are usually 3H (beta) or 125I (gamma)

4. Principle
• Involves competitive binding of radiolabelled antigen and unlabelled 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.
• Test samples of unlabeled antigen of unknown concentration are added in
progressively larger amounts.
• The antibody does not distinguish labeled from unlabeled antigen
• As the concentration of unlabeled antigen increases, more labeled antigen will be
displaced from the binding sites.

5. Technique
A mixture is prepared of radioactive Ag+ Ab ("First" antibody) against that antigen
Known amounts of unlabeled antigen are added to samples of the mixture
At increasing concentrations of unlabeled Ag, an increasing amount of radioactive
Ag is displaced from the antibody molecules
The Ab-Ag is separated from the free antigen in the supernatant fluid, and the
radioactivity of each is measured.
From these data, a standard binding curve can be drawn
The samples to be assayed (the unknowns) are run in parallel
After determining the ratio of bound to free antigen (cpm Bound/cpm Free) in each
unknown, the antigen concentrations can be read directly from the standard curve .

6. Radioimmunoassay: pros and cons
PROs:
• Versatility : using the same principle, almost any biomolecule can be assayed.
• Fast : (usually 2 days or less)
• Sensitive : (0.0006–0.006 µg antibody/ml)
• Large capacity : thousands of samples/day
• Specific : ( - antibody dependent)
CONs:
• Use of radioactivity: hazardous
• Expensive equipment (gamma or beta counter )

7. Applications

8. Introduction
 Levels of serum Prostatic Acid Phosphatase rises in Prostatic Cancer.
Samples:
1) Control- Human Prostatic Acid Phosphatase purified from prostatic fluid of
healthy males.
2) Antiserum- From female rabbits inoculated with purified PAP.
3) Sample for assay- Blood sample from patients

9. Methods:
1) RIA:
Polypropylene tubes coated with 0.5 ml of antiserum at appropriate dilution
Then tubes were incubated with 3 ml of 3% BSA in PBS
10µl of I125 labeled Antigen ( 2× 104 cpm~ 0.2ng) added n mixed
Incubation & washing
Known amount of unlabeled PAP added at increasing concentrations
Washes were given to remove free Ag.
Radioactivity measured and standard curve plotted
Serum concentration of unknown PAP calculated from standard curve

10. 2) Enzyme Assay:
Substrate – Paranitrophenyl phosphate
Enzyme - Patients Sample
Optical Density- 410 nm
Results:

11. REFERENCES
1) Andras G. Foti et al., Detection of Prostatic Cancer by Solid Phase Radioimmunoassay of
serum Prostatic Acid Phosphatase, The New England Journal of Medicine, Vol.297, Dec 22
1997, 1358-1361
2) Andras G. Foti et al., A Solid-Phase Radioimmunoassay for Human Prostatic Acid
Phosphatase, CANCER RESEARCH, Vol. 35, September 1975, 2446-2452.
3) Kuby Immunology, 5th Edition.

12. Thank
You