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

1. 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

11. Advantages of RIA
 The RIA can measure very minute concentration of
antigen even upto 10-12g 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.

12. 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.