1. RIA
Radioimmunoassay (RIA) is an in vitro technique used to measure concentrations of antigens (for example, hormone levels in the blood) without the need to use a bioassay.
The technique was developed by Berson and Yalow in 1960 to measure concentration of insulin in the blood.
Principle
In 1977, Rosalyn Sussman Yalow received the Nobel Prize in Medicine for the development of the RIA for insulin: the precise measurement of minute amounts of such a hormone was considered a breakthrough in endocrinology.
RIA are assays that are based on the measurement of radioactivity associated with immune complexes i.e antigen-antibody reaction to estimate a ligand. The reaction is as follows

2. Ag + Ag* + Ab AgAb + Ag*Ab + Ag + Ab
Where Ag= Antigen to be measured
Ag*= Radiolabeled Antigen
This process also induces the following mechanisms-
Unbound Ag* and Ag are washed out
Radioactivity of bound residue is measured
Antigen concentration is inversely related to radioactivity

3. Procedure
A mixture of radioactive antigen and antibody is prepared
+ unlabelled antigens into the mixture
Radioactive antigen is displaced by unlabeled antigen from the antibody
Antibody-antigen bound complex is separated from the antigen in supernatant fluid
Radioactivity of each is measured
A standard binding curve is found

4. Radioactive antigen
First antibody
Unlabeled antigen ( )
Radioactive antigen
Unlabeled antigen ( )
Precipitate (ag-ab complexes with anti-immunoglobulin (Second antibody)
Radioactivity of supernatant=free antigen
Radioactivity of ppt=bound antigen
Second antibody

5. Detection
The following substances are detected by RIA.
Hormones: insulin, GH, Thyroxin, Estrogen
Serum Protein: IgE antibodies
Some metabolites : cAMP, folic acid
Drugs: Digoxin, Digitoxin, Morphine
Microbial agents and antibiotics: Hepatitis B surface antigen in a donated blood

6. Advantages
High specificity: Immune reactions are highly specific
High sensitivity: Immune reactions are highly sensitive
Disadvantages
Radiation hazards
Requires specially trained person
Labs require special license to handle radioactive materials
Requires special arrangements for-
Requisition and storagetive material
Radioactive waste disposal

7. Both 125I and 131I emit gamma radiation that requires special counting equipment
The body can absorb iodine atoms (radioactive or not) in the TG where they are incorporated into thyroxin
Application
RIA is used to assay the plasma levels of
Different types of hormone
Digitoxin or digoxin in patients receiving these drugs
Certain abused drugs
It is used to assay the presence of hepatitis B surface antigen in donated blood
It is used to measure anti-DNA antibodies in systemic lupus erythematous

8. Although the RIA technique is extremely sensitive and extremely specific, it requires specialized equipment, but remains the least expensive method to perform such tests. It requires special precautions and licensing, since radioactive substances are used. Today it has been supplanted by the ELISA method, where the antigen-antibody reaction is measured using colorimetric signals instead of a radioactive signal. However, because of its robustness, consistent results and low price per test , RIA methods are again becoming popular.
The RAST test (radioallergosorbent test) is an example of radioimmunoassay. It is used to detect the causative allergen for an allergy.

9. Radioimmunoassays (RIA) are assays that are based on the measurement of radioactivity associated with immune complexes. In any particular test, the label may be on either the antigen or the antibody. Enzyme Linked Immunosorbent Assays (ELISA) are those that are based on the measurement of an enzymatic reaction associated with immune complexes. In any particular assay, the enzyme may be linked to either the antigen or the antibody

10. Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody as illustrated in Figure 2.
Affinity is the equilibrium constant that describes the antigen-antibody reaction as illustrated in Figure 3. Most antibodies have a high affinity for their antigens.

11. Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. In general, there is a high degree of specificity in antigen-antibody reactions. Antibodies can distinguish differences in:
The primary structure of an antigen
Isomeric forms of an antigen
Secondary and tertiary structure of an antigen

12. Avidity Reactions between multivalent antigens and multivalent antibodies are more stable and thus easier to detect.
Factors affecting measurement of antigen-antibody reactions
The only way that one knows that an antigen-antibody reaction has occurred is to have some means of directly or indirectly detecting the complexes formed between the antigen and antibody. The ease with which one can detect antigen-antibody reactions will depend on a number of factors.
Affinity The higher the affinity of the antibody for the antigen, the more stable will be the interaction. Thus, the ease with which one can detect the interaction is enhanced

13. Antigen to antibody ratio The ratio between the antigen and antibody influences the detection of antigen-antibody complexes because the size of the complexes formed is related to the concentration of the antigen and antibody. This is depicted in Figure 6.
Physical form of the antigen The physical form of the antigen influences how one detects its reaction with an antibody. If the antigen is a particulate, one generally looks for agglutination of the antigen by the antibody. If the antigen is soluble one generally looks for the precipitation of the antigen after the production of large insoluble antigen-antibody complexes.

14. To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two chemically bind to one another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added. This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody binding sites. As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter. Using known standards, a binding curve can then be generated which allows the amount of antigen in the patient's serum to be derived.radio immuno assay is a very important method in which presence of antigen antibody can be detected by using a radio active substance.

15. It was developed by Rosalyn Yalow and Solomon Aaron Berson in the 1950s.[1] In 1977, Rosalyn Sussman Yalow received the Nobel Prize in Medicine for the development of the RIA for insulin: the precise measurement of minute amounts of such a hormone was considered a breakthrough in endocrinology.
With this technique, separating bound from unbound antigen is crucial. Initially, the method of separation employed was the use of a second "anti-antibody" directed against the first for precipitation and centrifugation. The use of charcoal suspension for precipitation was extended but replaced later by Drs. Werner and Acebedo at Columbia University for RIA of T3 and T4.[2] An ultramicro RIA for human TSH was published in BBRC (1975) by Drs. Acebedo, Hayek et al.[3]

16. Radioactive antigen
First antibody
Unlabeled antigen ( )
Radioactive antigen
Unlabeled antigen ( )
Precipitate (ag-ab complexes with anti-immunoRadioactive antigen