RADIOIMMUNOASSAY EARLY

STAGES OF DETECTION OF
CANCER
Disease
Antigens are substances that cause the immune system to respond.
Carbohydrate antigen (CA) 19-9 is a type of antigen released by
pancreatic cancer cells. It can also be referred to as a tumor marker.
The CA 19-9 Radioimmunoassay (RIA) is a blood test that measures the
CA 19-9 level in the blood.
There are blood tests commercially available that may be able to detect
pancreatic cancer. A test measuring CA 19-9 cannot detect the presence
of pancreatic cancer by itself, but it can be used on a panel with other
biomarkers that can signal the presence of the disease. After diagnosis,
the CA 19-9 Radioimmunoassay (RIA) blood test can also be used for
some patients to watch the disease’s development.
Levels of CA 19-9 may be measured as part of a panel of biomarkers to
detect the disease early from a blood test. Patients may also get the test
after a confirmed pancreatic cancer diagnosis. A high CA 19-9 is usually
related to pancreatic cancer. But other cancers, like colorectal, lung and
gallbladder cancers, can also cause raised levels.

TEST INFORMATION
Radioimmunoassay (RIA) is an in vitro assay that measures the
presence of an antigen with very high sensitivity. Basically any biological
substance for which a specific antibody exists can be measured, even in
minute concentrations. The target antigen is labeled radioactively and
bound to its specific antibodies (a limited and known amount of the
specific antibody has to be added). A sample, for example a blood-
serum, is then added in order to initiate a competitive reaction of the
labeled antigens from the preparation, and the unlabeled antigens from
the serum-sample, with the specific antibodies.
The competition for the antibodies will release a certain amount of
labeled antigen. This amount is proportional to the ratio of labeled to
unlabeled antigen. A binding curve can then be generated which allows
the amount of antigen in the patient's serum to be derived. That means
that as the concentration of unlabeled antigen is increased, more of it
binds to the antibody, displacing the labeled variant. The bound antigens
are then separated from the unbound ones, and the radioactivity of the
free antigens remaining in the supernatant is measured. A binding curve
can be generated using a known standard, which allows the amount of
antigens in the patient's serum to be derived.

Radioimmunoassay is an old assay technique but it is still a widely used

assay and continues to offer distinct advantages in terms of simplicity
and sensitivity.

TEST PRINCIPLE
Antigens and antibodies bind specifically to form the Ag-Ab complex.
The antigen can be labeled or conjugated with radioisotopes. The
unlabeled antigens from the sample compete with radiolabeled antigens
to bind on paratopes of specific antibodies. The unlabeled antigens
replace labeled antigens that are already linked with the antibodies. The
unlabeled antigens when bind with antibodies, increases the amount of
free radiolabeled antigens in the solution. Hence the concentration of
free labeled antigens is directly proportional to the bound unlabeled
antigens.
It involves a combination of three principles.
An immune reaction i.e. antigen, antibody binding.
A competitive binding or competitive displacement reaction. (It gives
specificity)
Measurement of radio emission. (It gives sensitivity)

PROTOCAL
 Label 2 normal tubes.
 vortex calibrators, samples and controls and dispense 25 μl of
each
 Dispense 50 µl Biotin Anti-CA 19-9 in each tube.
  Incubate for 30 minutes at room temperature on a tube shaker
(400 rpm).
 Aspirate (or decant) the content of each tube (except total counts.
 Wash tubes with 2 ml Working Wash solution (except total
counts) and aspirate (or decant)
 Dispense 100 µl of 125Iodine labelled anti CA19-9 into each tube
 10. Incubate for 60 minutes at room temperature on a tube
shaker (400 rpm).
 Wash tubes with 2 ml Working Wash solution (except total
counts) and aspirate (or decant).
 Let the tubes stand upright for two minutes and aspirate the
remaining drop of liquid.
 Count tubes in a gamma counter for 60 seconds.

RESULTS

Calculate the mean of duplicate determinations. On semi logarithmic or

linear graph paper plot the c.p.m. (ordinate) for each calibrator against
the corresponding concentration of CA 19-9. The normal CA 19-9 range
in a healthy person is 0-37 units per millilitre. CA 19-9 levels can be
higher in patients with pancreatic cancer. Rising CA 19-9 values mean
the tumour is growing. If the values stay the same, the disease may be
stable. Decreasing CA 19-9 values may mean treatment is working and
the tumour or amount of cancer in the body is decreasing. A decline in
CA 19-9 levels after treatment followed by a rise later may suggest that
the tumour has come back or grown.