METHODOLOGY OF THE ASSAY
The methodology of the radioimmunoassay have been studied extensively and outlined in a sequential manner as follows :
1) Mix a fixed volume (fixed concentration ) of antiserum containing the specific antibody with a constant amount of radiolabelled antigen,
2) Incubate it for come specified duration at an appropriate temperature, usually + 4 °C,
3) A definite volume of the sample containing the hapten to be measured is added to the reaction test-tube,
4) The antibody reacts with both the radioactive and unlabelled hapten forming an antibody-radiolabelled antigen and antibody-unlabelled antigen complexes,
5) Since, both the radioactive and non-radioactive antigens (haptens) are more or less chemically and immunochemically the same, they will eventually compete for the limited number of antibody sites available ; thus, the amount of radioactivity that ultimately combines with the antibody will be an inverse function of the amount of unlabelled hapten competing for these sites,
6) The radioactivity falls because the unlabelled antigen dilutes it i.e., reducing the number of la-belled hapten combining with the antibody,
7) The counts obtained from the radioactivity are used to determine the hapten concentration in the sample, the interpretation being done on the standard curve, and
8) RIA is an exquisitely sensitive assay method that is capable of measuring with great accuracy (hapten) concentrations in nanograms and picograms utilizing very small volumes of the sample.
(i) In order to measure the radioactivity in the labelled hapten-antibody complex of the free hapten (labelled) a convenient means of separating these fractions is usually adopted,
(ii) The method of assaying the radioactivity of the bound and/or unbound fraction following separation, solely depends on the nature of the isotope and on the method utilized for the separation of the bound and unbound fractions,
(iii) Thus, one may actually determine either the antibody bound fraction or the unbound fraction routinely, but in the preliminary experiments it is always necessary to determine both these fractions, and compare them with a standard containing the total number of counts added in order to make sure that there are no losses unaccounted for,
(iv)The validity of RIA entirely depends upon the identical behaviour of standard and labelled substance unknown, and not on the identity of the labelled tracer and the unknown. Hence, the experimental condi-tions of incubation of standards and unknowns must be identical for any factors that might affect the extent of the immunochemical reaction, pH, ionic composition, protein content or any other substances of inter-est. However, these conditions may be tested conveniently and can be controlled effectively by preparing standards in hormone free plasma at the same dilution at which unknowns are assayed.