What is Radioimmunoassay ( RIA )

What is Radioimmunoassay  (RIA) ?

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Radioimmunoassay (RIA)

Introduction to                                  Radioimmunoassay :-

Radioimmunoassay (RIA) is an immunoassay that use radioactive molecules.
It involves the use of either antiserum or more usually Antigen labelled with radioisotopes such as 125-I. It is very sensitive and extremely specific method.
It is used to measure the concentration of substances such as Antibody and Antigen, usually used for measure Antigen concentration by use of Antibodies.
Ex. :- Measuring the Hormone level in blood.
It requires specialized equipments and requires special precautions and licensing.
The amount of radioactive label bound to antigen-antibody complex can be measured, and hence the concentration of antigen or antibody in a specimen can be determined.

History of                                            Radioimmunoassay :- 

This method is developed by Rosalyn Sussman Yelow , Roger Guillemin and Andrew Schally at the Veterans Administration Hospital, Bronx, New York. for the concentration of insulin in plasma.
It represented the first time that Hormone levels in blood could be detected by an invitro assay.
Dr. Yelow was awarded by Nobel prize for this achievement in 1977.

Principle :- 

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Radioimmunoassay (RIA) involves the separation of protein  ( from a mixture ) using the specificity of antigen-antibody  binding and quantitation using radioactivity.
RIA is based on the competition of radiolabelled, known antigen and unlabelled, unknown antigen for limited binding sites on known specific antibody in a solution or attached to a plate or tube (solid phase ).
RIA permits measurement of analytes up to picogram ( 10-12 ) quantities.
It has been used to measure a variety of serum proteins including Igs, Hormones, drugs, tumor markers and viral antigens.

Radioimmunoassay Method :- 

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Antigen-antibody reaction

To establish an assay, Antibody must be produced against the known antigen and the antigen radiolabelled e.g.:- with 125 I. 
A standard curve is established by adding fix amount of radiolabelled antigen, specific antibody and increasing concentrations of known unlabelled antigen to a series of tubes or wall in a plastic microtitre plate.
Following incubation, The amount of labelled antigen bound antibody
is determined after separation from free labelled antigen in a gamma spectrometer.
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Standard curve 

If test serum contains antigen for the antibody, then there is competition with labelled antigen.
This appears as reduction in the amount of labelled antigen bound in the complex.
The concentration of antigen in the sample can be determined from the standard curve.

Advantages :- 

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RIA is widely used  because of it's   high sensitivity. 
Using antibodies of great affinity, it is allow the detector a few the program ( 10-12 ) of antigen in a tube.
High specificity of antiserum send high specific sitting of essay.

RIA is a major tool in laboratories where it is used to assay plasma levels of :-

          - Hormones (most of human body)
          - Digitoxin or Digotoxin in patients receiving certain abused drugs.

To detect the presence of Hepatitis B surface antigen ( HBsAg ) in donated blood.
Anti DNA antibodies in systemic lupus erythematosus (SLE).

Application of                                    Radioimmunoassay :-

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  • Screening of donated Blood for evidence of viral contamination - 

- HIV -1 ( presence of Abs )
- Hepatitis C ( presence of Abs)
- Hepatitis B ( for both Abs and viral Ags )

  • Measuring Hormones levels :- 

LH ( luteinization hormone), TSH ,T3, T4 (for thyroid function )
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  • Infection Detection :- 

Sexually transmitted agents such as HIV, Syphilis, and chlamydia.
-Hepatitis B and C
-Toxoplasma gondii

  • Allergens detection in food and house dust :-  RAST  -

             RAST (Radioallergosorbent test) to detect specific IgE antibodies to suspected or known allergens.

  • Measuring the "rheumatoid factors " and other auto antibodies in autoimmune disease like lupus erythematosus. 
  • Measurement of toxins in contaminated foods.

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