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.
Note :
(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.
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