RIA (Radio Immuno Assay) is an immuno assay technique used for the detection of antigen (Ag) in a sample with the use of radio isotopes. It is an invitro antigen antibody interaction invented by Rosalyn Yalow and Salonin Berson in 1960 and at Veterans administration hospital,New York.
Principal: Antigens and antibodies bind specifically to form the Ag-Ab complex where antigen can be labelled or conjugated with radio isotops.The unlabelled Antigens from the simple complete with the radio labelled antigens to bind on paratops of specific antibodies.
The unlabelled antigens replace labelled antigens that are link with the antibodies. The unlabelled antigens when bind with antibodies,increases the amount of free radio labelled antigens in the solution. Hence the concentration of free labelled Antigens is Directly proportional to the bound unlabelled antigens.
Read: ELISA (Enzyme Linked Immuno Sorbent Assay )
REQUIREMENTS:
1.Radio labelled antigens(Ag labelled with isotopes like I-25 and tritium)
2.Unlabelled simple antigens called cold Antigens.
3.specific antibodies
4. Microtitre (96 wall microtitre plate)
5. washing buffer solution (1% triflouroacetic acid)
RIA Procedure
1) Specific antibodies of known concentration are fixed in the microtitre wall.
2) A known amount of hot antigen are added to the well.
3) washed carefully to remove unbound.
4) at this point, radioactivity of the wall will be maximum.
5) Unlabelled Antigen are added to the well.
6) the Unlabelled antigen will replace the radio level hot antigen and bind to the antibodies, freeing the hot Antigen
7)Carefull washing to remove the free hot Antigen.
8) The 0unity of radio level hot antigen measured by gamma counters. The intensity of radio activity is poporsonal to the concentration of antigen present patient sample.
Also read: Procedure of ELISA
Application:
1.Determination of very small quantities of Antigen and Antibody in the serum.
2.Used for quantification of hormones, drugs and other viral antigens.
3. Analyse nano molar and pico molar concentration of hormones in biological fluid.