Radioimmunoassay

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 3 H (beta ) or 125 I (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 . T est 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: U se of radioactivity : hazardous Expensive equipment (gamma or beta counter )

7. Applications

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

9. Methods: 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 10 µl of I 125 labeled Antigen ( 2× 10 4 cpm ~ 0.2ng) added n mixed Incubation & washing K nown 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 Andras G. Foti et al., Detection of Prostatic C ancer by Solid Phase Radioimmunoassay of serum Prostatic Acid Phosphatase, The New England Journal of Medicine, Vol.297, Dec 22 1997, 1358-1361 Andras G. Foti et al., A Solid-Phase Radioimmunoassay for Human Prostatic Acid Phosphatase, CANCER RESEARCH, Vol. 35, September 1975, 2446-2452. Kuby Immunology, 5th Edition.

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