RRA

1. Radioreceptor Assay
AISHWARYA BABU
M.SC. BIOTECHNOLOGY
SEMESTER 3

2. Competitive radioassay (radioreceptor assay)
Refers to a class of sensitive and versatile
analytic techniques that employ radionuclides in
vitro for the measurement of hormones and other
trace substances in biologic fluids.
All the various methods for in vitro radioassay
have in common a binding reagent (receptor)
which selectively binds the substance to be
measured (ligand).
A small amount of unlabelled ligand competes
with radioactively labelled ligand (radioligand) for
a limited number of binding sites on the receptor.
2

3. This radioassay technique has been given various names such as competitive
binding assay, radioligand assay, radioimmunoassay (when the binder is an
antibody), saturation analysis, displacement analysis and radiosteroassay.
Since all of the various methods have in common a receptor-ligand interaction,
and it is primarily the receptor which confers the sensitivity and specificity to the
assay procedure.
The conceptual basis of radioreceptor assay was developed in 1956 by Berson and
Yalow.
3

4. Basic Procedure
Step 1 : Incubation of the receptor with the substance to be
assayed together with a constant amount of that same
substance labelled with a radioactive nuclide (radioligand).
Incubation periods range from a fraction of an hour up to
several days, depending on the properties of the reagents of a
specific assay.
During the incubation period, the unlabelled ligand and the
radioligand will both bind to the receptor.
4
Receptor binding techniques involve the following steps:

5. However, the amount of receptor added is chosen so that its binding capacity is small
and will bind only a fraction of the available radioligand, even in the absence of any
additional unlabelled ligand.
Therefore, the ligand and the radioligand compete with each other for the limited
number of binding sites on the receptor.
Stated another way, increasing the concentrations of the unlabeled ligand in the
system will "dilute" the amount of radioligand being bound to the receptor, and so
reduce the amount of bound radioactivity.
5

6. Step 2. Isolation of the receptor fraction.
At the end of the incubation period, the amount of radioactivity
bound to the receptor will be inversely proportional to the
concentration of the substance to be assayed.
Referred to as the "separation step."
6

7. Step 3. Measurement of radioactivity bound to the isolated
receptor fraction.
7

8. Step 4. Translation of radioactivity measured into an estimate
of the concentration of the substance to be assayed.
This is performed by comparing the counts bound to the
receptor in the unknown sample versus counts recorded from
a series of standards made up with samples to which known
amounts of the ligand have been added.
The results of this standard series is most often plotted as a
standard curve giving the relationship between the
radioactivity measurement (on the y-axis) and the amount of
added ligand (x-axis).
8

9. Components
The development of a radioreceptor assay involves
the following prerequisites for the ligand to be
assayed:
It must be possible to label the ligand with a
radioactive nuclide;
It must be possible to produce a receptor with
relatively high affinity for the ligand;
Methods for isolating the receptor from the reaction
mixture must be available.
9

10. Radioactive Labelling
Some substances contain elements which have radioactive isotopes.
Radioactive isotopes of the most common biological elements, C, N, O, and H have either too short
or too long half-lives.
Very long half-lives like 14C or 3H, result in low specific radioactivity that reduces the sensitivity of a
radioreceptor assay to a degree which makes it impossible to measure many biological substances.
It is frequently necessary to introduce a radioactive isotope of an element which is not present in
the native molecule, if this can be achieved without altering the configuration of the native
molecule significantly.
10

11. Iodine isotopes 125l and 131l have been most useful since it is possible to iodinate most
proteins and polypeptides.
Both of these isotopes are gamma emitters with high enough specific activities and
decay rates to allow good sensitivity in a radioreceptor assay.
The half-lives of 125l (60 days) and 131l (8 days) are long enough to allow a practical shelf
life for labelled reagents.
The energy of their emitted radiation permits the use of crystal scintillation techniques
for efficient and simple radioactivity measurements.
11

12. Production of Receptor
Radioreceptor assays have been developed by taking the advantage of specific
binding properties of certain plasma proteins such as thyroxine binding globulin and
transcortin.
Receptors of tissue origin, either from the cytoplasm or from cell membranes have
been utilized (e.g., estrogen and gonadotrophins, cytoplasmic receptors, insulin and
growth hormone, membrane receptors).
The binding of hormone ligand to these specific tissue receptors is a part of the
process whereby the hormone exerts its biologic effect.
Thus, theoretically, when a tissue receptor is used in a radioreceptor assay, the assay
will measure biologic activity
12

13. Separation Step
Conventional protein separation techniques like ammonium sulphate precipitation,
electrophoresis or gel chromatography may be used for the separation of receptor-bound
and free radioligand.
Other separation techniques have included paper chromatography or a combination of
chromatography and electrophoresis called chromatoelectrophoresis, ethanol precipitation,
absorption to a solid phase such as charcoal, chalk or silica.
Some receptors of tissue origin are insoluble and may be isolated by simple centrifugation.
13

14. Characteristics of Radioassay
The most prominent feature of radioreceptor assays was their extreme sensitivity.
Radioreceptor assays are now used for substances occurring in concentrations
ranging from a few picograms up to hundreds of nanograms.
Corresponding to molar concentrations in the order of 10-11 to 10-9 M.
Another feature - Specificity.
It has been possible to raise antisera which may differentiate between two large
polypeptides which differ only by one or two amino acids.
14

15. The general quality of a receptor assay is thus mainly determined by the quality of the receptor.
Specificity is a function of how accurately the receptor can differentiate one substance from another.
While the sensitivity is determined by the avidity of binding between the ligand and the receptor.
Technical Performance
Radioreceptor assays require only a few analytical steps and usually no primary extraction or
purification procedures.
Therefore, technical performance consists primarily of repetitive pipetting.
A large number of samples can be conveniently analysed by one technician who is often assisted by
equipment for automated pipetting.
Reliable counting equipment with capacity to" handle up to 1,000 samples automatically has made the
radioactivity measurement one of the easiest parts of the assay.
Treatment of the large amount of counting data varies from simple manual reading of the values off the
standard curve to the use of a computer which automatically determines the appropriate value.
15

16. Application of Radioreceptor Assays
The specific binding properties of antibodies have allowed the initial determination of trace
quantities of polypeptide hormones, such as insulin, glucagon, growth hormones and
gonadotrophin.
Assays were developed for small polypeptides like ACTH and angiotensin and even
dipeptides like triiodothyronine.
The specific binding properties of plasma proteins have been exploited to measure
corticosteroids, thyroxine and sex hormones.
Cell binding sites that are being used in radioreceptor assays include membrane receptors
for insulin, ACTH and catecholamines, and cytoplasmic receptors for estrogen.
16

17. 17

18. 18