Radioimmunoassay: History, Principle, Applications:
In this article, we are going to discuss another immunological Technique which is known as radioimmunoassay this technique is used to determine the concentration of antigen present in the sample. This technique is so sensitive that it can detect the 0.001 micrograms of antigen per ml of sample.
What is Radioimmunoassay (RIA) :
- Radioimmunoassay (RIA) is one of the most important immunological techniques for detecting antigens and antibodies to quantify the substances to be tested.
- RIA is also called competitive saturation analysis.
- RIA makes it possible to measure substances in the body that were difficult to measure in the past.
- In 1960, American chemists R.S. Yalow and S.A. Berson proposed this method, and Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977.
- In the early 1980s, more than 300 biologically active substances were determined by this method.
- Radioisotopes commonly used to label antigens include ³H, ¹²⁵I, ¹³¹I, etc.
- Commonly used coupling reagents are carbodiimides, isoxazole salts, alkyl chloroformates, diisocyanates, and imitates.
- This technology has the characteristics of high sensitivity, specificity, and accuracy and can be widely used in endocrinology, drug and toxic substance analysis, virus and tumor-related antigen determination, etc.
- In the 1960s, Dr. Rosalyn Yalow and Dr. S. A. Berson jointly published the “Principle of Competitive Protein Binding”, and developed radioimmunoassay technology based on this principle.
- These two scientists uses this technique first time to determine the level of insulin anti-insulin complexes in diabetics.
- For the discovery and development of radioimmunoassay technology, Yalow win Nobel Prize in Medicine in 1977.
- RIA has rapidly promoted the vigorous progress of modern medicine and basic research, coupled with the successful research of commercial kits, the operation process is simple and easy, making biomedical diagnosis more convenient.
- In fact, currently, widely used enzyme immunoassay (EIA) or enzyme luminescence assay are derived from this radioimmunoassay technology.
Principle of RIA :
- The principle of RIA is based on the competitive binding of radiolabeled and non-labeled antigens with antibodies. That is why RIA is also called competitive radio saturation assay.
- When we mixed *Ag is an isotope-labeled antigen, which has the same immune activity as the unlabeled antigen, with the antibody at a concentration that saturates the antigen-binding sites of the antibody.
- The two kinds of antigen (Rediolabeled *Ag and unlabeled Ag) combine with antibody (ab) competitively to form Ag-Ab or Ag-Ab complexes, which reach after a certain reaction time.
- If an effective separation method is used to separate *Ag-Ab and Ag-Ab complex from *Ag and Ag and measure the radioactivity of both.
Types of Radioimmunoassay :
There аre twо different methоds оf RIА thаt аre соmmоnly used fоr drug deteсtiоn in immunological reactions, dоuble-аntibоdy RIА аnd соаted-tube RIА.
(a) Double-Antibody RIA –
In double-antibody RIA, a second antibody is added to facilitate precipitation of the bound primary antibody. Once the primary/secondary antibody-antigen complex precipitates, the unbound labeled drug can be easily removed.
(b) Coated-tube RIA –
In coated-tube RIA, the primary antibody is coated on the inside of each tube. The unbound labeled drug can be easily removed by pouring off the supernatant.
If we wish to do this we must have three things with us
- Anti-monoclonal antibodies: against the antigen this antibody will bind to an antigen present in the sample these antibodies are generally prepared by Hybridoma technology.
- Radiolabeled antigen: means some part of this antigen is made up of radioisotopes. so that they can emit radiation like Gamma or beta. Generally, heavy isotopes of ³H or ¹²⁵I are used for the reaction.
- Unlabeled antigens
Process of radioimmunoassay :
- a microtiter plate is taken and then it is coated with anti A monoclonal antibodies
- After this radiolabeled antigens are added to the well but in the excess quantity. (Now what will happen if the concentration is kept high. since there is a large number of antigens. All the antigen-binding sites of antibodies will be saturated by the antigens, no antibody will be left free. as you can see some radiolabeled antigens are left unbound that are not bound to the antibodies.)
- Now the Unbound Antibodies are removed with help of washing. (In this situation there is a well coated with the antibodies and all the antibodies are bound to the radiolabel antigens therefore let us assume the radioactivity of antibodies or presence on the antibodies is 100% since all the antibodies have radiolabeled antigen.
- After this, a very small amount of unlabeled antigen is added to the well. We added 1ng antigens in well. (What is the effect of this Adition will create the competition between radiolabeled and unlabeled antigen for binding antibodies and this competition is proportional to the concentration of unlabeled antigen added to the well. since we have added only one 1ng of antigen competition will be less and various radiolabelled antigens will be removed from the antibody and of course, that place will be taken by unlabelled antigen.
- Now-Again washes the well to remove unbound antigens whether it is labeled or unlabeled but unbound antigens. (Now what is the situation this time number of radiolabeled antigens bound to the antibodies is decreased to the addition of unlabeled antigens and hence the radioactivity present on the antibodies is not hundred percent.
- So we can say when label antigens were not added to the well that is 0ng, radioactivity was 100% and when one 1ng of unlabeled antigens were added it decreased due to the competition and let us assume it is 90%.
Advantages/ Applications of Radioimmunoassay:
- Due to the development of radioimmunoassay technology, physicians can detect subtle changes in early disease, and have a better understanding of disease treatment and tracking, especially in the application of tumor markers, which makes early detection and early treatment easier.
- The main advantages of RIA are very simple to test and it is highly sensitive.
- It is an extremely sensitive assay as it can measure antigen up to nanogram quantities.
- This method is used to determine insulin, growth hormone, parathyroid hormone, angiotensin, prolactin, luteinizing hormone, follicle-stimulating hormone, prostaglandin, etc.
- It is a highly specific radioimmunoassay used to measure the concentration of antigens, hormone levels by the use of antibodies directed against the antigens.
- Radioimmunoassay (RIA) can detect Narcotic Drugs such as Heroin & Morphine, chlorpromazine, phenytoin, gentamicin, digoxin, theophylline, etc.
- RIA applications also include the determination of rheumatoid factor, complement and anti-food antigen antibodies, thyroglobulin antibodies, etc.
- In oncology for the determination of carcinoembryonic antigen, folic acid, vitamin B12, plasminogen, fibrinogen, etc.
- In endocrinology, RIA is used to identify, diagnose, and study the physiology and pharmacology of hormones.
- It is a useful technology to study the mechanism of hormone-receptor binding.
- Determination of immunoglobulin G, immunoglobulin E, and anti-deoxyribonucleic acid antibodies in clinical immunology.
- It is a relatively rapid and simple method to detect drug poisoning and drug metabolism.
Disadvantages of RIA (Radioimmunoassay) :
- Radiation hazardous.
- Require special arrangements for the storage of radioactive material.
- The high cost of waste disposal.
- Sometimes cross-reaction, the false-positive reaction occurs,
- Lengthy counting time
- There are some difficulties in the automation of this assay.
- The reaction time is long due to the use of a highly diluted reagent.
- Radioisotopes are costly and have Lengthy counting time.
- cannot inactivate degrading enzymes and salts.
- pH sometimes affects the results, etc.