Radioimmunoassay

Radioimmunoassay (RIA) or radioimmunological assays include such methods of radioisotope microanalysis, that their basis of is the immunochemical reaction of an antigen with a specific antibody (Ab), and which is carried out in vitro in the presence of a suitable radiolabeled compound as a radioindicator, which allows the quantification of the assay based on the determination of the activity distribution.

History
The method by which the blood insulin was first measured in vitro was developed in the 1950s in the USA. It was the first ever quantitative determination of the level of the hormone in the blood. For this discovery, Rosalyn Sussman Yalow received the Nobel Prize in Medicine in 1977. By being able to accurately measure the level of insulin, the treatment of diabetes mellitus has moved up a significant step forward.

The principle of the method
It is a competitive immunoreaction, i.e. the labeled antigen competes with the unlabeled antigen for binding sites on the antibody, which is present in a limited amount in the reaction mixture. In this case, the determined substance is an unlabeled antigen, another antigen is labeled, for example, with a radioactive isotope of iodine ( 125 I, 131 I) for protein antigens, or with tritium or 14C for low molecular weight substances. The result of the reaction is the formation of two complexes: labeled antigen-antibody (Ag*-Ab) and unlabeled antigen-antibody (Ag-Ab). The amount of the labeled complex (Ag*-Ab) is inversely proportional to the amount of the determined antigen, i.e. the more the determined substance is in the sample, the smaller the amount of the labeled complex will be and the smaller the resulting signal will be. This can be simply explained by the fact that the labeled antigens do not bind to the antibody due to the lack of binding sites to occupy the unlabeled antigen. From the above findings, it also leads to that free forms of Ag and Ag* are also found in the reaction mixture. The total reactivity (T) is thus divided into two fractions – bound (B) and free (F), while the following applies: T = B + F.

Method prodecedure
For the quantitative determination of certain substance in the examined sample, it is necessary to create a calibration curve that reflects the depedence of the resulting signal on the known concentration of the given substance. We prepare the calibration curve from the so-called standards. Controls, where we know the amount of radioactivity in advance, are an integral part of the determination.

Solid-phase RIA (in tubes)
 * 1) Invidual standards and unknown samples are pipetted into test tubes coated with a specific antibody. The control samples are pipetted into the uncoated tubes.
 * 2) Add the same amount of radioindicator (Ag*) to each test tube.
 * 3) Mix and let it incubate.
 * 4) After a sufficiently long time, we aspirate the reaction mixture.
 * 5) We measure bound (B) and free (F) radioactivity on a gamma counter.

Note: Separation of the immunocomplex can also be done in other ways, e.g. using electrophoresis, ion exchange chromatography , etc.

=== Examples of the use of RIA in practice ===


 * endocrinology (hormone levels in the blood)
 * digitoxin or digoxin in of patients taking these drugs
 * toxicology: evidence of the presence of drugs
 * blood transfusion: presence of hepatitis B surface antigen (HBsAg) in donated blood
 * imunology: anti-DNA antibodies in systemic lupus erythematosus (SLE)

Advantages and disadvantages of RIA
The main advantages of this method are high sensitivity and the possibility of automation. The disadvantage is the necessity of an intermediate separation stage, the expensive equipment required to perform this method and, last but not least, the risks associated with handling the radioactive substance.

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