Radioimmunoassay (RIA)

Background

Radioimmunoassay (RIA) is an immunoassay technology created by Yalow and Berson in 1959 that can quantify specific antigens in samples. RIA is an immunological detection technique that uses radionuclide labeled antigen. It combines the sensitivity of radionuclide and the specificity of immune response, and is an ultra-micro determination method. The commonly used radioisotopes in RIA are I¹²⁵ and I¹³¹. Because I¹²⁵ has the advantages of moderate half-life, high nuclide abundance, active chemical properties, easy labeling, and small impact on the immune activity of the labeled substance, etc., it is widely used in RIA. RIA has the advantages of high sensitivity, strong specificity, simple operation, and wide application range. It can be used to determine hormones, peptides, proteins, vitamins, drugs, etc.

The Principle of RIA

The radioactive labeled antigen (Ag*) and the unlabeled test antigen (Ag) in the sample competitively bind with the quantitative specific complementary antibody (Ab). Then add second antibodies specific to the complementary antibodies to bind to the complementary antibodies and form a complex that participates at the bottom of the well, thereby separating the complementary antibodies from the solution. Subsequently, centrifugation forms a precipitate containing radioactive antigens, sample antigens, complementary antibodies and secondary antibodies. Then the concentration of the target antigen in the sample can be determined by measuring the amount of conjugate (Ag*-Ab) and according to the standard curve. The target antigen content in the sample is inversely proportional to the radiation obtained.

RIA Procedure

1. Sample processing. Use RIA buffer to prepare sample dilutions containing the antigen to be tested, prepare standard solutions at the same time (set 6 gradients for the standard solution, and set two duplicate tubes for each gradient), and mark each test tube. In addition to samples and standard solutions, contains tubes for non-specific binding (NSB), total binding (TB), and total count (TC).
2. Add 100 µL of standard solutions to the standard tubes, and add 100 µL of sample dilutions to the sample tubes, then add 100 µL of RIA buffer to the NSB tubes and TB tubes.
3. Except TC tubes and NSB tubes, add 100 µL of complementary antibody solution to all test tubes.
4. Prepare a mixture consisting of RIA buffer and radioactively labeled antigen (tracer). Add 100 µL of tracer to each tube, containing sample, standard, NSB, TB and TC tubes. Vortex each tube briefly and incubate overnight at 4°C.
5. Add 100 µL of the secondary antibody dilution solution, vortex each tube briefly and incubate at 4℃ for 30 min.
6. Centrifuge at 6000g for 30 min at 4°C (except for the TC tubes), remove the supernatant and aspirate the remaining liquid.
7. Measure and calculate the amount of radioactivity in each tube with a gamma counter and software.

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Reference:

1. Alhabbab, R.Y. (2018) Radioimmunoassay (RIA). In: Basic Serological Testing. Techniques in Life Science and Biomedicine for the Non-Expert. Springer, Cham.