Clinical immunology

Lab.1

Immunology is a branch of biomedical science that covers the study of all aspects
of the immune system in all organisms.
immunity is the balanced state of having adequate biological defenses to
fight infection, disease, or other unwanted biological invasion, while having
adequate tolerance to avoid allergy, and autoimmune diseases.
Immunereaction:The reaction resulting from the recognition and binding of an antigen by its specific antibody or by a previously sensitized lymphocyte.
Antigens: are any substances that are capable, under appropriate conditions, of
inducing the formation of antibodies and reacting specifically with the antibodies so
produced. These antigenic molecules may have several antigenic determinants, called
epitopes, and each epitope can bind with a specific antibody. Thus, a single antigen
can bind to many different antibodies with different binding sites.

Antibody(Immunoglobulin):any of numerous Y shaped protein molecules produced

by B cells as aprimary immune defense, each molecule and its clones having a unique
binding site that can combine with the complementary site of a foreignantigen, as on
a virus or bacterium, thereby disabling the antigen andsignaling other immune defens
es.An immunoglobulins test is done to measure the level of antibodies in blood.
The five major types of antibodies (Immunoglobulin) are:

IgA. IgA antibodies are found in areas of the body such the nose, breathing passages,
digestive tract, ears, eyes, and vagina. IgA antibodies protect body surfaces that are
exposed to outside foreign substances. This type of antibody is also found in saliva,
tears, and blood. About 10% to 15% of the antibodies present in the body are IgA
antibodies. A small number of people do not make IgA antibodies.
IgG. IgG antibodies are found in all body fluids. They are the smallest but most
common antibody (75% to 80%) of all the antibodies in the body. IgG antibodies are
very important in fighting bacterial and viral infections. IgG antibodies are the only
type of antibody that can cross the placenta in a pregnant woman to help protect her
baby (fetus).
IgM. IgM antibodies are the largest antibody. They are found in blood and lymph
fluid and are the first type of antibody made in response to an infection. They also
cause other immune system cells to destroy foreign substances. IgM antibodies are
about 5% to 10% of all the antibodies in the body.
IgE. IgE antibodies are found in the lungs, skin, and mucous membranes. They cause
the body to react against foreign substances such as pollen, fungus spores, and animal
dander. They are involved in allergic reactions to milk, some medicines, and some
poisons. IgE antibody levels are often high in people with allergies.
IgD. IgD antibodies are found in small amounts in the tissues that line the belly or
chest. How they work is not clear.
The levels of each type of antibody can give your doctor information about the cause
of a medical problem.
Autoantibodies:
The critical function of the immune system is to discriminate self from non-self.
Tolerance against self-antigens is a highly regulated process and, in order to maintain
it, the immune system must be able to distinguish self-reactive lymphocytes as they
develop. The presence of autoantibodies is the consequence of breakdown of
tolerance and, although they are an important serological feature of autoimmune
diseases, their presence is not exclusive of these conditions. Antibodies against selfantigens are also found in cancer, during massive tissue damage and even in healthy

subjects. Natural autoantibodies provide immediate protection against infection and

also prevent inflammation by facilitating the clearance of oxidized lipids, oxidized
proteins, and apoptotic cells; their role in development of autoimmunity is still
unclear. Detection of serum autoantibodies in clinical practice has become more
available to clinicians worldwide while providing a powerful diagnostic tool.
An autoantibody is an antibody (a type of protein) produced by the immune
system that is directed against one or more of the individual's own proteins. Many
autoimmune diseases are caused by such autoantibodies.

Immunology

Lab.2

Agglutination reactions produce visible aggregates of antibody-antigen complexes

when antibodies or antigens are conjugated to a carrier.
Agglutination reactions apply to particulate test antigens that have been conjugated
to a carrier. The carrier could be artificial (such as latex or charcoal particles) or
biological (such as red blood cells). These conjugated particles are reacted with
patient serum presumably containing antibodies. The endpoint of the test is the
observation of clumps resulting from that antigen-antibody complex formation.
The quality of the result is determined by the time of incubation with the antibody
source, amount and avidity of the antigen conjugated to the carrier, and conditions of
the test environment (e.g., pH and protein concentration).
Various methods of agglutination are used in diagnostic immunology and these
include latex agglutination, direct bacterial agglutination, and hemagglutination.
In latex agglutination, many antibody molecules are bound to latex particles,
which increases the number of antigen-binding sites. If an antigen is present in
a test specimen, it will bind to the antibody and form visible, cross-linked
aggregates. Latex agglutination can also be performed with the antigen
conjugated to the particles for testing the presence of antibodies in a serum
specimen.
Direct bacterial agglutination uses whole pathogens as a source of antigen. It
measures the antibody level produced by a host infected with that pathogen.
The binding of antibodies to surface antigens on the bacteria results in visible
clumps.

 Hemagglutination uses erythrocytes as the biological carriers of bacterial

antigens, and purified polysaccharides or proteins for determining the presence
of corresponding antibodies in a specimen .
Agglutination tests are easy to perform and in some cases are the most sensitive
tests currently available. These tests have a wide range of applications in the
clinical diagnosis of noninfectious immune disorders and infectious disease.

Precipitation assays
Precipitation assays are performed in semi-solid media such as agar or agarose
where antibodies and antigens can diffuse toward one another and form a visible line
of precipitation.
There are several precipitation methods applied in the diagnostic laboratory. These
include single, double, and electroimmunodiffusion.
The most widely used gold standard precipitation methods are Ouchterlony test and
Mancini test.

precipitin Any antibody which reacts with an antigen to form a precipitate.

Precipitation reactions are based on the interaction of antibodies and antigens. They
are based on two soluble reactants that come together to make one insoluble product,
the precipitate . These reactions depend on the formation of lattices (cross-links)
when antigen and antibody exist in optimal proportions.
Precipitation reactions differ from agglutination reactions in the size and solubility of
the antigen and sensitivity. Antigens are soluble molecules and larger in size in
precipitation reactions.
There are several precipitation methods applied in clinical laboratory for
the diagnosis of disease. These can be performed in semisolid media such as agar or
agarose, or non-gel support media such as cellulose acetate.
Precipitation methods include double immunodiffusion (qualitative gel technique that
determines the relationship between antigen and antibody), radial immunodiffusion
(semi-quantitation of proteins by gel diffusion using antibody incorporated in agar),
and electroimmunodiffusion (variation of the double immunodiffusion method
reaction that uses an electric current to enhance the mobility of the reactants toward
each other).

Precipitation reactions are less sensitive than agglutination reactions but remain gold
standard serological techniques.
The most commonly used serologic precipitation reactions are:
Ouchterlony test (based on double immunodiffusion and named after the Swedish
physician who invented it). In the double immunodiffusion technique, three basic
reaction patterns result from the relationship of antigens and antibodies. These
patterns are identity, non-identity, and partial identity.
Mancini method (based on single radial immunodiffusion).
The Mancini method results in precipitin ring formation on a thin agarose layer. The
diameter of the ring correlates with the concentration of proteins in the precipitin.

Mancini method

Ouchterlony test