| Radioimmunoassay was developed by yellow & berson
to measure insulin in human plasma. The technique has been used
extensively to measure small amount of proteins & hormones in
fluid. Advances in these techniques have largely been responsible
for great strides made in last 10-15 years in endocrinology
research. However because of rather cumbersome technique, need of
special lab, instruments& trained persons & risk of radioactive
rays exposure, These techniques have not found much place in busy
diagnostic microbiological laboratory.
Two following basic techniques are used:
- Competitive RIA
- Excess reagent RIA
Competitive RIA
This technique is of common use. It is performed
as follows:
A constant amount of antibody (known) to the
antigen to be assayed is added to a series of tubes. A small
amount of Radio labeled antigen plus increasing amounts of
unlabelled antigen are then added to the tubes. After a period of
incubation , the antibody bound ( labeled & unlabelled) antigen is
separated from the free or unbound antigen by one of the several
methods such as precipitation by 50% saturated ammonium
sulfate, or by anti-globulin. The amount of radioactivity of
precipitate (bound labelled antigen) and of supernatant (
free labelled antigen) are determined.
A graph is plotted to shoe relationship of amount
of unlabelled antigen added to the ratio of bound& free labelled
antigen. From this curve the concentration of unknown antigen in
test sample can be determined by determining the ratio of bound &
free labelled antigen in test procedure performed by adding test
sample to the tube containing antibody & labelled antigen in
amounts used for preparing graph.
This technique has been used to measure levels of
many antigens, drugs, hormones etc in human body fluids.
Excess
reagent RIA
In excess reagent technique the antibody is
usually labelled and used in excess concentration. Analyte is
measured by determining the radioactivity in the conjugate, Which
is separated as described in competitive RIA.
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Solid
Phase RIA
Polystyrene microlitre plates or beads are used as
solid phase. Wells or beads are coated with specific antibodies to
the antigen to be assayed. These are treated with specific
antibodies to the antigen to be assayed. These are treated with
test sample and then with radio labelled antibodies in known but
excess amount. The absorbed radioactivity is measured & compared
with similar activity in similar test with negative control.
samples with residual counts 2.1 times or greater than that on
negative control are considered positive. Similarly known
antibodies can be detected by using antigen coated wells
DETECTION
OF ANTIBODIES BY Solid Phase RIA
-
Competitive binding RIA
→
The solid phase is coated with known antigen & then treated
with mixture of test sample & known amount of labelled antibody.
The radioactivity of bound labelled antibody is determined. It is
inversely proportional to the amount of antibody in the test
sample.
-
Blocking
or Inhibition RIA
→
In this method the test sample is first incubated with
known quantity of antigen, then the mixture is transferred to
solid phase coated with antibodies & treated with labelled
antibodies. The amount of labelled antibodies will be less if the
test sample contains less antibodies compared with negative
control. a reduction in counts of 40% or more is considered as
positive.
-
Detection of specific IgM
The solid phase is first coated with anti- IgM,
Then treated with test serum, Than known antigen & finally with
radio labelled antibody. The absorbed radioactivity is
proportional to the specific IgM in the serum. |
