Radioimmunoassay (RIA)

Radioimmunoassay (RIA) is an in vitro assay that measures the presence of an antigen with
very high sensitivity. Usually presence of any antigen and antibody can be determined and
measured even in minute concentration. RIA is one of the most sensitive & specific methods of
immune assays available and the first technique to analyze upto picomolar concentration of any
biologically substances.  Furthermore, as the name indicates, it is an immunological assay to
analyze any antigen or anti-body in the patient’s serum to diagnose the disease.

History: The technique was developed on 1960 by S. A. Berson and Rosalyn Yalow and
Rosalyn R. Yalow who received the Nobel Prize for it in 1977. It was the first assay technique to
determine the presence of hormone level in blood using invitro assay.

Principle: Basically Radioimmunoassay (RIA) depends upon three principles which have made
it most specific and sensitive technique with the sensitivity range is 0.0006–0.006 µg
antibody/ml. immune reaction, a competitive binding and measurement of radio emission assay
are the three principles which in sum execute the RIA.

The classical RIA methods are based on the principle of competitive binding. In this method, an
unlabeled antigen competes with a radiolabeled antigen for binding to an antibody with the
appropriate specificity. Thus, when mixtures of radiolabeled and unlabeled antigen are incubated
with the corresponding antibody, the amount of free (not bound to antibody) radiolabeled antigen
is directly proportional to the quantity of unlabeled antigen in the mixture.

It involves a combination of three principles.

1. An immune reaction i.e. antigen, antibody binding.

2. A competitive binding or competitive displacement reaction. (It gives specificity)
3. Measurement of radio emission. (It gives sensitivity)

Immune Reaction:

When a foreign biological substance enters into the body bloodstream through a non-oral route,
the body recognizes the specific chemistry on the surface of foreign substance as antigen and
produces specific antibodies against the antigen so as nullify the effects and keep the body safe.
The antibodies are produced by the body’s immune system so, it is an immune reaction. Here the
antibodies or antigens bind move due to chemical influence. This is different from principle of
electrophoresis where proteins are separated due to charge..

Competitive Binding or competitive displacement reaction:

 This is a phenomenon wherein when there are two antigens that can bind to the same antibody,
the antigen with more concentration binds extensively with the limited antibody displacing
others. So here in the experiment, a radiolabelled antigen is allowed to bind to high-affinity
antibody. Then when the patient serum is added unlabeled antigens in it start binding to the
antibody displacing the labeled antigen.

Procedure:

 Needed substances and equipment: Specific antiserum to the antigen to be measured,

availability of a radioactive labeled form of the antigen, a method in which the antibody-
bound tracer can be separated from the unbound tracer, an instrument to count radioactivity.
 RIA involves the separation of protein from the mixture using the specific reaction of
antigen-antibody reaction and quantitation using radioactivity.
 Radioactivity: 125-I labels are usually applied although other isotopes such as C14 and
H3 have also been used. Usually iodination are used to label the antigen by chloramine-T or
peroxidase methods and then separating the radio-labeled antigen from free-isotope by gel-
filtration or HPLC. The target antigen is labeled radioactively and bound to its specific
antibodies (a limited and known amount of the specific antibody has to be added).
 During competitive binding and displacement, there are two antigens which can bind to
same antibody, the antigen with more concentration binds extensively with the limited
antibody displacing other.
 Antigen–antibody complexes are precipitated either by crosslinking with a second
antibody or by means of the addition of reagents that promote the precipitation of antigen–
antibody complexes. Counting radioactivity in the precipitates allows the determination of the
amount of radiolabeled antigen precipitated with the antibody.
 After the washing to remove the unbound antigens, RIA utilizes a radioactive label which
emits radiation that can be measured with a beta or gamma counter.

In RIA the sequential steps followed are:

1) Mix a fixed concentration of antiserum containingspecific antibody with a constant of

radiolabelled antigen.

2) Incubate it for specified duration at an appropriate temperature usually +4°c.

3)A definite volume of sample containing unlabelled antigen to be measured is added to the
reaction test tube.

4) The antibody reacts with both radioactive & unlabelled antigen forming an ab-radiolabelled
antigen complexes.
5) Both radioactive & unlabelled antigens are more or less same immunochemically; they will
compete for limited number of antibody sites available.

6) The radio activity falls because the unlabelled antigen dilutesit.i.e) reducing the no of labelled
antigen combining with antibody.

7) The counts obtained from the radioactivity are used to determine the unlabelled antigen
concentration in the sample, the interpretation being done on the standard curve.

Calibration curve:

A standard curve can be constructed by plotting the percentage of antibody-bound radiolabeled

antigen against known concentrations of a standardized unlabeled antigen as shown in figure.
Also, the curve allows determining the unknown antigen concentration directly from the standard
curve.
 Uses of Radioimmunoassay (RIA):

RIA has revolutionized research and clinical practice specially in blood banking, diagnosis of
allergy and endocrinology. Furthermore it is used to:

1. The test can be used to determine very small quantities (e.g. nanogram) of antigens and
antibodies in the serum.
2. The test is used for quantitation of hormones, drugs, HBsAg, and other viral antigens.
3. Analyze nanomolar and picomolar concentrations of hormones in biological fluids.

Limitations of Radioimmunoassay (RIA):

1. The expenses of equipment and reagents along with hazards if preparing and handling the
radioactive antigens.
2. Short shelf-life of radiolabeled compounds
3. Requirements of special counter for radioisotopes.
4. The problems associated with the disposal of radioactive waste.

Advantages:

1. It is a saturation analysis as active reagent added in smaller quantity than that of analyte

2. It is indirect method of analysis

3. It is structurally specific as antigen: antibody reactions are highly specific.

4. Radio immuno assay is very sensitive technique used to measure concentrations of antigen
without the need to use a bioassay. It can measure one trillionth (10-12) of a gram of material per
milliliter of blood.
Disadvantages of RIA:

1. Prolonged reaction time (in days) as a consequence highly diluted reagent is used.
Radioactive Iodine used in is not a cheap reagent.
2. Possible health hazards due to handling of radioisotopes.
3. All the reagents must be added precisely.
4. Limited assay range.
5. Lack of direct linear relationship between analyte concentration and signal response.
6. Difficulty of automation.
7. Lengthy counting time. 

Applications:

1.  Evaluation of enzyme immunoassay and radioimmunoassay methods for the

measurement of plasma oxytocin
2. Significance of prostate specific antigen in prostate cancer patients and in non cancerous
prostatic disease patients.
3. Estradiol measurement in translational studies of breast cancer. Plasma estrogen
measurement with use of radioimmunoassay has been instrumental in the development of
aromatase inhibitors for endocrine therapy of postmenopausal breast cancer.
4. Refinement of Zalcitabine pharmaco kinetics.