Radio immunoassay

The technique of RIA was developed by Rosalyn Yallow. FIGURE

Insulin was the first substance thus quantitated by RIA.
Nowadays, hormones, growth factors, tumor markers
cytokines, bacterial antigens, and any other biological
substances could be quantitated accurately by the ria
method. The specificity of antibody and the sensitivity of
radioactivity are combined in this technique. Suppose,
blood level of thyroxin (T4) is to be assayed. The T4
hormone is the antigen(Ag) in this case. It is made to react
with a specific antibody(Ab). A constant amount of
isotope-labelled hormone, constant amount of antibody and
variable quantities of unlabelled hormone are taken in
different tubes.
Tube No. of No. No. of Labeled Radio-
No. molecules molecules molecules molecul activit
of anti- of labeled of es in y in is a competition between the unlabelled hormone (antigen)
body hormone unlabelled supernat precipi present in the biological specimens and the added labeled
hormone ant tate antigen to combine with the antibody. The more the
added activity
1 100 100 0 0 100 unlabelled antigen, less of the labeled antigen will combine
2 100 100 100 50 50 with the antibody. The antigen-antibody reaction is
3 100 100 300 75 25 allowed to take place for a definite period of time. At the
4 100 100 900 90 10
5 100 100 Patient’s A Z end of the incubation period, the tube will contain free and
serum bound antigen. The bound and free forms are separated by
protein precipitating agents such as polyethylene glycol or
In tube A, the labeled hormone molecules are combined a second antibody. The radioactivity of the bound form in
with the antibody molecule; so there is no radioactivity in the precipitate is measured. A series of standard tubes
the supernatant. In B, equal quantity of unlabelled hormone containing known but varying concentration of the pure
is added, when labeled and unlabelled antigen molecules antigen are taken along with the unknown biological
compete for the antibody. Thus, half of radioactivity is in specimen. The level of the hormone in the specimen can be
the supernatant and half in the precipitate. The obtained from a calibration curve prepared from the
displacement of labeled antigen is proportional to the measured radioactivity of the known standards.
unlabelled antigen in the system. A series of test tubes are ADVANTAGE OF RIA:- By this method, microgram and
taken, in which constant quantity of antibody, constant picogram quantities of substances could be analysed. The
quantity of labeled antigen and different but known radioisotopes commonly used for labeling the antigen I125.
quantities of unlabelled antigen are added. After a few DISADVANTAGES OF RIA:-1. Since radio-istopes are
hours of incubation, a precipitating agent is added, when used, there are stringent laws: and only approved
antigen-antibody complex, being high molecular weight laboratories could take up the assay.
substance, is precipitated. The radioactivity in the 2. Half life I125 isotopes is about 60days: iodinated antigen
precipitate is inversely related to the unlabelled antigen should be used within a few months. The shelf-life of the
added. The values of the radioactivity in the precipitate are reagent is short.
shown in table. In the same series of test tubes, patients NONISOTOPIC IMMUNOASSAYS:-They have the
serum may be added as the source for unlabelled hormone. advantage that there is no radiation hazard. So the test
The radioactivity in the precipitate is plotted in this graph. could be doe in any clinical laboratory. The shelf-life is
In the same series of test tubes, patients serum may be also more. The ELISA technique is more simple and less
added as the source for unlabelled hormone. The time consuming than RIA. Instead of the radio label, an
radioactivity in the precipitate is plotted in this graph at the enzyme I is tagged. The antibody is usually conjugated
Y-axis, when the corresponding value in the X-axis will with the enzyme. The enzyme labels commonly used are
give the actual quantity of hormone present in that sample. alkaline phosphate (ALP) and horse radish peroxidase
GRAPH
There (HRP).
CHROMATOGRAPHY:-The term is derived from the
Greek word chroma, meaning colour. The method was first
employed by Tswett, a botanist in 1903, for the separation
of plant pigments using a column of of alumina. Nowadays
HPLC is used to separate almost all biological substances,
including proteins, carbohydrates, lipids and nuclei acids.
1.ADSORPTION CHROMATOGRAPHY:- In this
technique the separation is based on differences in
adsorption at the surface of a solid stationary medium. The
common adsorbing substances used are alumina, silicates
or silica gel. These are packed into columns and the
mixture of proteins to be separated is applied in a solvent
on the top of the column. The components get adsorbed on
the column of adsorbent with different affinity. The
fractions slowly move down; the most weakly held fraction
moves fastest; followed by others, according to the order of
tightness in adsorption. The eluent from the column is
collected as small equal fractions and the concentrations of
each is measured, in each fractions.
2. PARTITION CHROMATOGRAPHY:- This
technique was developed by martin and Synge in 1941. this
includes different types depending on the phases between
which the components are partitioned, e.g. solid-liquid,
liquid-liquid, gas-liquid, etc. This is commonly used for
the separation of mixtures of amino acids and