What Is The Principle Behind Radioimmunoassay?

WHY SCIENCE IS FOCUSING ON RADIOIMMUNOASSAY?

• There is hardly any other specific immunoassay technique, used for quantitative detection of antigens or haptens, which is as sensitive as a radioimmunoassay (at a concentration of <0.01 μg/mL), thus making it an essential tool in biomedical, clinical practices and also for drug discovery, monitoring, and food testing.

• It is also possible to detect as low as a few picograms of an analyte when using antibodies of high affinity

• A specialized radioimmunoassay named RAST is also used to measure the amount of IgE antibody that reacts with a known antigen of known concentration.

• Most commonly, RIA is used to evaluate proteins, hormones, vitamins, microbial antigens, or drugs in the serum.

WHAT IS IMMUNOASSAY?

• It is a bioanalytical test to determine the presence of small quantities (nanogram) of specific antigens and specific antibodies in the serum, blood, or in other fluids.

• When Elisa immunoassays are used to assay the presence of an antigen in the fluid system, then the test contains the specific antibody as part of the whole detection system. When used for detecting the presence of antibody, then the test system contains specific antigen in it.

PRINCIPLE OF IMMUNOASSAY?

This method uses the specificity of an antigen and antibody binding/reaction to detect the total aggregate in the biological sample.

WHAT IS LABELED IMMUNOASSAY?

Before starting with the principle of RIA, we must know about the labelled immunoassay. RIA is a classification of it.

Here we use a variety of labels to detect the antigen, antibody, or analytes present.

The crucial components of a labelled immunoassay:

• an analyte
• an antibody
• a label

WHAT IS RIA?

• A radioimmunoassay uses radiolabelled molecules( antibodies) in a gradual formation of the immune compound to detect and quantitate the amount of antigen (analyte) present in the sample.

• It was first described in 1960 for the measurement of endogenous plasma insulin by Solomon Berson and Rosalyn Yalow of the Veterans Administration Hospital in New York.

PRINCIPLE OF RADIOIMMUNOASSAY?

• It completely works on the process named “competitive binding” where the antigen (tracer) will be labelled radioactively and which competes with a non-radioactive antigen for an endless number of receptor binding sites of an antibody.

• When unlabelled antigen from samples and a fixed amount of labelled antigen both are allowed to incubate or react with a limiting amount of the corresponding antibody, decreasing amounts of tracer are bound to the antibody as the number of unlabelled antigen increases.

• Thus in simple words, Radiolabelled antigen (“tracer”) added to an antibody specific to the antigen leads to the formation of an antigen-antibody complex. Unlabelled antigens from a sample or standard solution can also bind antibodies, leading to an unlabelled antigen-antibody complex.

• The amount of radiolabelled antigen (tracer) is held constant. Increasing amounts of unlabeled antigen in the sample will compete with the tracer for binding to the antibody, leading to a more unlabelled antigen-antibody complex.

• Now the amount of free (not bound to antibody) radiolabelled antigen is directly proportional to the quantity of unlabelled antigen in the mixture.

Therefore, to sum up, the principle in 3 points, radioimmunoassay is based on:

1. an immune reaction which is — antigen-antibody binding
2. competitive binding which gives specificity
3. quantification of radio emission, which gives sensitivity.

Now let us discuss all 3 points in brief.

1. IMMUNE REACTION-

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

2. COMPETITIVE BINDING-

• It can also be called a displacement reaction. This whole phenomenon was described in the starting of principles as it being the actual root.
• To explain in short again, we can say- when two antigens 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 a high-affinity antibody. Then when the patient serum is added unlabelled antigens, it starts binding to the antibody displacing the labelled antigen.

3. QUANTIFICATION OF RADIO EMISSION

• After the incubation is over, and washing is done to remove any unbound antigens. Then radio emission of the antigen-antibody complex is taken, the gamma rays from radiolabelled antigen are measured.
• Antigen-antibody complexes are precipitated either by crosslinking with a second antibody or utilizing the addition of reagents that promote the precipitation of antigen-antibody complexes.
• Counting radioactivity in the precipitates allows the determination of the amount of radiolabelled antigen precipitated with the antibody.
• A standard curve is constructed by plotting the percentage of antibody-bound radiolabelled antigen against known concentrations of a standardized unlabelled antigen, and the concentrations of antigen in patient samples are extrapolated from that curve.

Limitations of RIA-

1. Costly equipment and reagents.
2. Radiolabelled compounds have a short shelf life.
3. Problems associated with the disposal of radioactive waste.

Advantages of RIA-

1. You do not need the samples or substances in higher quantities and concentrations, even a minute level of sample size is accepted.
2. Most specific and sensitive immunoassay.