Radio immuno assay (priya)

Basics of Radio Immunoassay- Discovery, Procedure, Advantages and Disadvantages

1. RADIO IMMUNO ASSAY METHODS AND USES By M.Priya dharshana

2. INTRODUCTION Radio immunoassay is a technique to test and measure the concentration of the antigens by using radiolabelled antibodies This method is also known as the Binder ligand assay method.

3. Discovery It was first developed in 1959 by Berson and Yallow for the measurement of insulin in plasma For this discovery they were awarded the nobel prize in 1977.

4. REAGENTS USED IN RIA A tracer(beta emitter or gamma emitter) A binder(antibody) Sample serum Separating system Gamma counter

5. PROCEDURE Sample antigen is injected into a mouse and then its blood is taken after 4 hours. The blood is centrifuged to remove cells and large proteins. The remaining antiserum is used as the primary antibody. Now a known quantity of target antigen is labelled with the radioactive isotope 3H or 125I The antibody and the labelled antigen are mixed in a buffer at suitable pH.

6. Contd … The mixture is incubated at a suitable temperature The radiolabeled antigen binds to the binding sites of the antibody.30-50% of the radiolabelled antigen binds to the antibody. To this unlabelled sample antigen is added This results in the competition between the radiolabelled antigen and the sample antigen for the limited number of binding sites on the antibody.

7. Contd … The amount of radiolabbeled antigen bound to the antibody decreases as the concentration of the unlabelled antigen increases. To this reaction mixture second antibody is added.The sample antigen is injected into a rabbit and its blood is taken after 4 hrs. The blood is centrifuged to get the antiserum. This antiserum is secondary antibody The secondary antibody precipitates the antibody bound labelled antigen. The mixture is now centrifuged to isolate the precipitate that contains only the bound labelled antigen

8. Contd.. The antibody bound radiolabbeled antigen and free radiolabelled antigens are counted using the gamma counter

9. Contd.. A standard curve is generated using a set of labelled standards with known concentations . The amount of antigen in the test sample is calculated using the standard curve

10. Advantages of RIA The RIA can measure very minute concentration of antigen even upto 10 -12 g in a litre of sample. It is an indirect method of analysis Early cancer detection Measurement of growth hormone levels Blood bank screening Narcotic(drug) detection Diagnosis of peptic ulcers.

11. Dis advantages of RIA: Prolonged reaction time. Radioactive iodine used is not a cheaper reagent. Possible health hazards due to handling of radioisotopes. Lengthy counting time. All the reagents must be added precisely.

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