specific binding proteins (γ-globulins) which exhibit only a limited reactivity with other immunogens
even if they are closely similar in structure. Analytes with relative molecular masses of 6000 or greater
will usually produce the desired response themselves, but smaller molecules (haptens) may need to be
attached to a suitable protein before injection into the animal. A binding agent thus produced will
normally show sufficient specificity for the original analyte to provide the basis of an RIA method.
Once the level of antibody in the blood of the animal is high enough (6–12 weeks), blood is taken from
the animal and the serum, together with the antibodies, separated. Further separation is not needed and
the binding agent is used in this form. Serum containing antibodies thus produced is often known as
antiserum. Undoubtedly the production of a suitable antiserum is the most difficult stage in the
development of an RIA procedure, requiring specialist biochemical knowledge and skill. Subsequent
stages of the analysis are usually straightforward and a range of RIA reagents is now available in kit
form. Typical procedures involve the following stages:
(1) Mixing samples or standards in solution with a measured amount of the labelled analyte and a
measured substoichiometric amount of antiserum.
(2) Adjustment of solution conditions followed by incubation (e.g. at 2– 4 °C for 1 1/2 hours) to ensure
maximum and reproducible reaction.
(3) Separation of bound and free analyte. This is often done by adsorption of the free analyte on
activated charcoal or precipitation of the protein bound fraction with ammonium sulphate.
(4) Measurement of the ratio of radioactivity in the bound and free fractions.
The activity ratio A may be related to the amount of analyte X in the sample by
where Y and Z are, respectively, the amount of labelled analyte added and the amount equivalent to the
antiserum added (i.e. bound). However, in practice it is advisable to construct a suitable calibration
graph because of the danger of cross-reactions and frequent lack of linearity. The steps are summarized
diagrammatically in Figure 10.16 and typical calibration graphs (dose response curves) in Figure 10.17.
Applications of RIA
The determination of small amounts of organic compounds in complex matrices has long been a
difficult analytical problem. If separation procedures are used they frequently need to be complex and
are time consuming. By using specific binding reagents, RIA methods avoid such difficulties and
facilitate the analysis of many compounds at picogram levels