Radioimmunoassay (RIA) 27th October 2021 – Tags: ,

What is radioimmunoassay (RIA)?

Radioimmunoassay (RIA) is a type of immunoassay, or radioimmunometric method, that is based on the specific formation of antigen-antibody (Ag-Ac) complexes. which gives it great specificity combined with the sensitivity of radiological methods (radioactive isotopes).

Radioimmunoassay (RIA) consists of a laboratory technique of clinical analysis. It uses radioactive isotopes, is in vitro, i.e. blood is taken from the patient and tested for substances.

The target antigen is radioactively labelled and binds to its specific antibodies (a limited and known amount of the specific antibody must be added). A sample, e.g. blood serum, is then added to initiate a competitive reaction of the labelled antigens in the preparation, and the unlabelled antigens in the serum sample, with the specific antibodies.

Competition for the antibodies will release a certain amount of labelled antigen. This amount is proportional to the ratio of labelled to unlabelled antigen. A binding curve can then be generated which allows the amount of antigen in the patient’s serum to be deduced.

It is a technique that was born in the 60s and 70s, leading to a great advance in endocrinology. It is a very sensitive technique, measuring small quantities of blood.

What is the principle of the Radioimmunoassay?

Radioimmunoassay (RIA) is based on the principle of all immunoassays, which is the recognition of an antigen present in a sample by antibodies directed against this antigen.

This means that as the concentration of unlabelled antigen increases, more of the unlabelled antigen binds to the antibody, displacing the labelled variant. The bound antigens are then separated from the unbound antigens and the radioactivity of the free antigens remaining in the supernatant is measured. A binding curve can be generated using a known standard, allowing the amount of antigens in the patient’s serum to be derived.

Radioimmunoassay is an old assay technique, but it is still a widely used assay and still offers clear advantages in terms of simplicity and sensitivity.

Radioimmunoassay RIA

Substances and equipment needed:

  • Antiserum specific to the antigen to be measured
  • Availability of a radioactive labelled form of the antigen
  • A method wherein the tracer bound to the antibody can be separated from the unbound tracer
  • An instrument for counting radioactivity

Radioactivity:

125-I labels are usually applied, although other isotopes such as C14 and H3 have also been used. High activity (125-I) radiolabelled antigen is usually prepared by iodination of the pure antigen at its tyrosine residue(s) by chloramine-T or peroxidase methods and then separation of the radiolabelled antigen from the free isotope by gel filtration or HPLC. Other important components of the RIA are the antigen-specific antibody and the pure antigen for use as a standard or calibrator.

Separation techniques:

Double antibody, charcoal, cellulose, chromatography or solid phase techniques are applied to separate bound and free radiolabelled antigen. The most commonly used is the double antibody technique combined with polyethylene. The bound or free fraction is counted in a gamma counter.

At the same time, a calibration or standard curve is generated with samples of known concentrations of the unlabelled standards. The amount of antigen in an unknown sample can be calculated from this curve.

Sensibility

Sensitivity can be improved by decreasing the amount of radiolabelled antigen and/or antibody. Sensitivity can also be improved by a so-called non-equilibrium incubation. In this case, the radiolabelled antigen is added after the initial incubation of antigen and antibody.

Solution of problems

The antibody should be specific for the antigen under investigation (other antigens should not cross-react with the antibody). If any cross-reactivity is observed, the selection of a different antibody or purification of the antibody from the cross-reactive antigen by affinity chromatography is advised.