Procedure:

It involves a radiolabelled antigen (Ag*) along with inactive antigen (Ag) analyte of known concentrations (standards) and an antibody (Ab)-the reagent.  An important point to note is the requirement of an efficient and specific separation procedure for the complex Ag-Ab free from antigen.  Various steps to be followed are:

Antibody (Ab): It is the key reagent in RIA and efficiency of assay depends on the amount of Ab used. These molecules come as immunoglobulins with high molecular weight of ~150,000 Daltons. These are prepared by injecting Ag into laboratory animals such as rabbit, guinea pig, goat, sheep, chicken, monkey, etc. The antigen against which antibodies are required, is emulsified with a mineral oil and injected into the animal. Secondary injections or booster doses are given after 3-4 weeks interval. Normally good quality antibodies are produced after 3 to 4 such injections. Afterwards blood of immunized animals is collected and screened for antibody response. The serum, a white yellow liquid left after removal of red blood cells, is separated and assayed for antibody content and their specificity checked. It is essential to have pure antibodies because any impurity will show cross reactivity and it will overestimate the antigen concentration.