APPLICATION OF RADIOIMMUNOASSAY IN CHEMICAL PATHOLOGY
WHAT IS IMMUNOASSAY?
Immunoassay is a chemical test used to detect or quantify a specific substance, the analyte, in
blood or other body fluid samples using an immunological reaction. Immunoassays are highly
sensitive and specific. Their high specificity results from the use of antibodies and purified
antigens as reagents.
An antibody is a protein (immunoglobulin) produced by B-lymphocytes (immune cells) in
response to stimulation by an antigen.
Immunoassays measure the formation of antibody-antigen complexes and detect them via an
indicator reaction. High sensitivity is achieved by indicator system (e.g enzyme label) the results
in amplification of measured product.
Immunoassays may be qualitative (positive or negative) or quantitative (amount measured). An
example of a qualitative assay is an immunoassay test of pregnancy. Pregnancy tests detect the
presence of human chorionic gonadotropin in urine or serum. Highly purified antibodies can
detect pregnancy within two days of fertilization. Quantitative immunoassays are performed by
measuring the signal produced by the indicator reaction. This same test for pregnancy can be
made into a quantitative assay of HCG by measuring the concentration of product formed.
IMMUNE REACTION
When foreign biological substance enters into body blood stream through non oral route, body
recognizes the specific chemistry on surface of the foreign substance as antigen and produces
specific antibodies against the antigen so as to nullify the effects and keep the body safe. The
antibodies are produced by body immune reaction. Here the antibodies or antigens bind move
due to chemical influence.
WHAT IS RADIOIMMUNOASSAY?
Radioimmunoassay (RIA) is a sensitive method of measuring very small amount of substance in
blood. Radioactive version of a substance, or isotopes of the substance, are mixed with
antibodies and inserted in a sample of the patient’s blood. The same non-radioactive substance
in blood takes the place of the isotope in antibodies, thus leaving the radioactive substance
free.
The amount of free isotopes is then measured to see how much of the original substance was in
the blood. This isotopic measuring method developed in 1959 by two Americans, biophysicist
Rosalyn Yalow (1921-) and physician A. Berson (1918-1972).
HISTORY OF RADIOIMMUNOASSAY
Yalow and Berson developed the first radioisotopic technique to study blood volume and iodine
metabolism. They later adapted the method to study how the body uses hormones, particularly
insulin, which regulates sugar levels in the blood. The researchers proved that type II (adult
onset) diabetes is caused by the inefficient use of insulin.
In 1959 Yalow and Berson perfected their measurement technique and named it
radioimmunoassay (RIA). It is extremely sensitive. It can measure one trillionth of gram of
material per milliliter of blood. Because of the small sample required for measurement, RIA
quickly became a standard laboratory tool.
THE PRINCIPLE OF RADIOIMMUNOASSAY
This method is highly sensitive and it can be used to determine substances present in the body
fluids, IgD, IgE and hormones, in minuteamounts. The requirements are a supply of purified
radiolabelled antigen of the kind it is desired to test for, and its specific antibody. Measured
quantities of these two are then incubated together, alone (as a control), and with added test
serum. If there is antigen present in the test serum it consumes some of the antibody, and less
is available to react with the radiolabelled antigen. At the end of the test there is then more
free radiolabelled antigen in the test sample than in the control sample.
It involves three principle which make it more sensitive and specific than other immune
assays.
1. An immune reaction i.e. antigen, antibody binding.
2. A competitive binding or competitive displacement reaction. (it gives specificity)
3. Measurement of radio emission. (it gives sensitivity)
PRECAUTIONS TO BE TAKEN BEFORE THE TEST IS BEING CARRIED OUT
Blood samples are collected by vein puncture with needle. It is not necessary to restrict fluids or
food prior to collection. Blood samples should be collected in a plain bottle/ tube containing no
additive. Random urine samples are acceptable for drug assays, however, 24 hours urine
samples are preferred for hormones and other substances which show diurnal or pulse
variation.
Special safety precautions must be observed when performing RIA methods. Radioactive
isotopes are used by RIA test to label antigens or antibodies. Pregnant females should not work
in an area where RIA tests are being performed. Personnel handling isotope reagents must
wear badges which monitor their exposure to radiation. Special sinks and waste disposal
containers are required for disposal of radioactive waste.
PROCEDURE FOR CARRING OUT RADIOIMMUNOASSAY
1. The labeled antigen is mixed with antibody at a concentration that saturates the
antigen-binding sites of the antibody.
2. Then test samples of unlabeled antigen of unknown concentration are added in
progressively larger amounts.
3. The antibody does not distinguish labeled from unlabeled antigen, so the two kinds of
antigen compete for available binding sites on the antibody. As the concentration of
unlabeled antigen increases, more labeled antigen will be displaced from the binding
sites.
4. The decrease in the amount of radiolabeled antigen bound to specific antibody in the
presence of the test sample is measured in order to determine the amount of antigen
present in the test sample.
5. The antigen is generally labeled with gamma-emitting isotopes such as I125, but beta-
emitting isotopes such as tritium (3H) are also routinely used as labels.
6. The radiolabeled antigen is part of the assay mixture; the test sample may be a complex
mixture, such as serum or other body fluids, that contains the unlabeled antigen.
The procedure requires small amount of sample and can be conducted in small 96-well
microtiter plates; hence this procedure is suitable to determine the concentration of a
particular antigen in large numbers of samples. For example, a microtiter RIA can be used to
screen for the presence of hepatitis B virus. RIA screening of donor blood has sharply reduced
the incidence of hepatitis B infection in recipients of blood transfusion.
Radioimmunoassay is not widely used like elisa in health care because it is time consuming and
also very expensive.
APPLICATIONS OF RADIOIMMUNOASSAY
RIA has many applications including:
1. Narcotics (drug) detection
2. Early detection of cancer
3. Measurement of growth hormone levels
4. Diagnosis and treatment of peptic ulcers
5. Research with brain chemicals called neurotransmitters
6. Detection of antibodies for inhalant allergens
7. Detection of allergy diagnosis
ADVANTAGES AND DISADVANTAGES OF RADIOIMMUNOASSAY
RIA is a very sensitive and specific method for the assay of various antigens and antibodies.
Substances such as hormones or drugs, which can be antigenic in laboratory animals and
produce specific antibody can be assayed by this method. With increasing use of
monoclonalantibodies, RIA is proving to be very useful. The major disadvantages of RIA are the
use of radioactive compounds which have a limited shelf-life and need special staff training for
handling them. Counting radioactive emission requires expensive equipment. If the radiation is
not well monitored, it can be a potential health hazard.
Summary
Radioimmunoassay is a labelling technique used for the detection of antibody, or more
commonly, antigen. It uses radioactive label or tracer to assess the concentration of biological
molecules. Tritium (H3), I131 or I125 are commonly used as tracers.
RIA makes use of the principle of competitive binding between radiolabelled and unlabeled
molecules of antigen to bind with a high affinity, specific antibody. The amount of unlabeled
antigen present in the specimen is measured by its competitive effect on the labeled antigen for
limited antibody sites.
REFRENCES
1. http://www.surgeryencyclopedia.com/Fi-la/immunoassay-tests.html
2. http://encyclopedia2.thefreedictionary.com/radioimmunoassays
3. http://www.rajaha.com/radio-immuno-assay-principle-procedure-ria/
4. http://www.discoveriesinmedicine.com/Ni-Ra/Radioimmunoassay-RIA.html
5. http://www.ncbi.nlm.nih.gov/m/pubmed/9293309/
6. Medical laboratory science: theory and practice by J OCHEI and A KOLHATKAR
BABCOCK UNIVERSITY
ILISAN-REMO
OGUN-STATE.
DEPARTMENT OF MEDICAL LABORATORY SCIENCE
AN ASSIGNMENT IN IMMUNOLOGY AND IMMUNOCHEMISTRY (MLSC 414)
ON
AN ESSAY ON APPLICATION OF RADIOIMMUNOASSAY IN CHEMICAL
PATHOLOGY
BY
JOHN HANNATU SENCHI
MATRIC NUMBER
12/3732
SUBMITTED TO
MRS ADEJUMO E.N
ON
2ND MARCH 2015
BABCOCK UNIVERSITY
ILISAN REMO
OGUN STATE
IMMUNOLOGY AND IMMUNOCHEMISTRY
COURSE CODE: MLSC 414
TOPIC:
AN ESSAY ON TH APPLICATION OF RAIOIMMUNOASSAY IN
CHEMICAL PATHOLOGY
SUBMITTED BY:
JOHN HANNATU SENCHI
TO
MRS ADEJUMO E.N
DEPARTMENT:
MEDICAL LABORATORY SCIENCE
LEVEL:
400
DATE
2ND MARCH 2015
