RADIOIMMUNOASSAY AND BLOOD SEROLOGY

Immunoassay?

This is a biochemical analysis, qualitative or quantitative, that measures the presence or concentration
of micro or macromolecules in a solution through the use of an antibody or an antigen.

Radioimmunoassay(RIA) is an in vitro form of immunoassay that measures the presence of an antigen

or antibody with very high sensitivity using radio-labelled molecules in a stepwise formation of immune
complexes. A radioisotope is attached to an antigen of interest and bound with its complementary
antibody. Then a sample with the antigen to be measured is added. It competes with the radioactive
antigen, kicks it out of the binding spot and replaces it. After washing away unbound antigens the
radioactivity of the sample is measured. The amount of radioactive signal is inversely related to the
amount of target antigen.

Principle of radioimmunoassay

The basic principle of radioimmunoassay is competitive binding, where a radioactive antigen ("tracer")
competes with a non-radioactive antigen for a fixed number of antibody or receptor binding sites. When
unlabelled antigen from standards or samples and a fixed amount of tracer (labelled antigen) are
allowed to react with a constant and limiting amount of antibody, decreasing amounts of tracer are
bound to the antibody as the amount of unlabelled antigen is increased. This principle works based on
the presence of a fixed number of antibodies with the antigen added being the variable parameter.

How it works

Take for example, that a patient’s insulin is to be measured using RIA. First, known amounts of
antibodies and radioisotope-tagged insulin are mixed. These combine chemically to form complexes.
Next, a small amount of the patient’s blood is added. The insulin in the blood displaces some of the
tagged insulin from the complexes formed thus creating a free insulin-tagged insulin ratio which is
measured with isotope detectors. The patient’s insulin levels can then be calculated from the values
obtained.
History

Radioimmunoassay was developed by Rosalyn Yalow and Solomon A. Berson  at the Veterans
Administration Hospital in the Bronx, New York in 1960. RIA was used to study the metabolism of insulin
after its administration to diabetic patients. It earned Dr Yalow a Nobel prize in medicine in 1977.

Reagents and Instrumentation

Radioisotopes : Beta emitters (³H and ¹⁴C) and gamma emitters (¹² 5C)

Binder/Antibody

Separating medium : May be physical (filtration, sieving etc) or chemical (ammonium sulphate, ethanol
etc. solutions)

Analyte (in pure form) : serum, protein, plasma etc

Isotope detector / Gamma counter

Advantages of RIA

- It is very specific. Antigens antibody complexes only form between specific antigens and
antibodies.
- It is very sensitive and can measure minute quantities of antigen material per millimeters of
blood.
- It provides very accurate results.

Disadvantages of RIA

- Radioactive iodine is expensive.

- Possible health hazards due to mishandling of radioisotopes.
- Difficulty of operation.
- Limited assay range.

Uses of RIA

- Blood bank screening for Hepatitis virus

- Measurement of hormone levels; growth hormone, insulin etc.
- Diagnosis and treatment of peptic ulcers
 - Cancer detection and treatment
- Observation and analysis of neurotransmitter

Blood serology

Blood serology test is any laboratory used to detect the presence and amount of antibodies in blood
serum. These tests focus on detecting proteins made by the body’s immune system.

Presence of antigens provoke the immune system to produce antibodies specific to the type of antigen it
encounters. These antibodies are particles that attach to the antigens and deactivate them. Blood
serology tests identify the type of antibodies and antigens that are in your blood sample, and identify
the type of infection present.

Types of serologic tests

Agglutination assay : An agglutination assay shows whether antibodies exposed to certain antigens will
cause particle clumping.

Precipitation assay : A precipitation test shows whether the antigens are similar by measuring for the
presence of antibody in body fluids.

Complement fixation test : Used to check for presence of either specific antigen or antibody in patient
serum based on whether complement fixation occurs.

Enzyme-linked immunosorbent assay (ELISA) : This assay uses a solid-phase enzyme immunoassay (EIA)

to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed
against the protein to be measured.

History

Dr Jules Bordet (1870-1961) is generally regarded as the founding father of modern serology. He was a
Belgian immunologist, bacteriologist and physician who received the Nobel prize for Medicine in 1919
for his discovery of factors in blood serum that destroy bacteria. He discovered antibodies as the key
player in bacteriolysis that occurred in animals already immune to the specific bacteria and the role of
complement system in bacteria cell wall destruction. He also discovered and characterized agglutination.

He developed the complement-fixation test in 1904 with Octave Gengou, his brother-in-law.

Dr Geoffrey Tovey (1916-2001) was a English physician who performed early work on the typing of red
cells and their antigens, white blood cells (Human Lymphocyte Antigens or HLAs), and the transfusion of
platelets and later stem cells in the treatment of leukaemia.

How it’s done

Since different antigens will elicit the production of different specific antibodies, blood serology tests
vary for different antigens and the test done is mostly determined by the diagnosing doctor.

Blood sample is retrieved from patients from the vein with a syringe or tip of fingers with a lancet. This
sample is tagged and sent to the laboratory for testing.

If Brucellosis or Typhoid fever is to be tested for, the Widal test is employed. Bacteria that cause typhoid
fever(e.g. Salmonella) is mixed with the serum sample obtained from the patients which contains
antibodies. 2-mercaptoethanol is often added to this test as it denatures the IgM class of antibodies
without much effect on the IgG class. This is important to distinguish between recent antibody response
(IgM) and old response from a former infection (IgG).

7-14 days is needed for the rise in antibody levels (seroconversion) that would correctly diagnose
infection. The Widal test is positive if TO antigen titre is 1:160 in an active infection. If testing shows no
antibodies, then it can be concluded that there is no infection.

Other tests include the Kastle-Meyer test used in forensic serology for chemical identification of blood,
Landsteiner’s agglutination test, Weil-Felix test for the diagnosis of Rickettsia infection.

Advantages of blood serology

- It is very accurate in illness and auto-immune disorder diagnosis.

- It is relatively inexpensive test to carry out.

Disadvantages of blood serology test

- Because of the window period needed for seroconversion (7-14 days or more), diagnosis is slow.
- Chemical oxidants may provide false-positive results before testing is concluded in Kastle-Meyer
test.
Uses of serology blood tests

- Serology blood tests help to diagnose patients with certain immune deficiencies associated with
the lack of antibodies, such as X-linked agammaglobulinemia.
- It is used to determine an individual’s blood type.
- Forensic serology is heavily used in crime fighting to determine individual by body fluid e.g.
linking a rapist to a semen sample.
- It is used in the diagnosis of illnesses like Brucellosis, measles, rubella, HIV, syphilis, amebiasis,
fungal infections etc.