MCB303 - Salem University

MCB 303: Immunology
3 Credits
Organs and Tissues of the Immune System
Antigen and Antibody interactions /Reactions
Structure of antigens, antigenic determinants
Structure and classification of immunoglobulin and
Mechanism and theory of antibody formation.
Role of lymphoid tissues and thymus in immune
Complement System.
Toxin and Antitoxin Reactions
Diagnostic immunology, Vaccines, effectors systems of
parasite killing and nature of resistance in plants.
• The Human body is continuously in contact to microorganism
(pathogenic and non pathogenic).
• The organism present in our environment such as water, air, food
, soil etc.
• The pathogenic organisms are capable of causing infections that
leads to diseases.
• These pathogens are generally not part of Normal floral of the
• The pathogens include Viruses, Bacteria and Fungi.
• Despite this contact with pathogens, vertebrates particularly
man do not frequently get infected thereby resulting into
• Generally, therefore man has highly sophisticated mechanism of
developing resistance to varieties of disease causing pathogens.
• These defense mechanisms are known as
Resistance factors.
• These factors serve to suppress the growth
and spread of the pathogenic micro-organism.
• The defence system can be generally divided
into 2 broad groups.
• Specific Host Defences: which are directed
towards individual organism; is also known as
Immune Response.
• The Non-Specific Defences: Mechanism which
can also be referred to as the Natural Host
• Are brought about by the immune system, the
immune system becomes activated when foreign
molecules collectively known as Antigen
• When a foreign body such as pathogens are introduced
into the body system, the individual cells of the
immune system are stimulated to produce - Antibody
or Immunoglobulins
• This is achieved by process known as Humoral
• Once the antibody is produced it interacts specifically
with the antigen that stimulate each production in
order to neutralize the effect.
• In addition the antigen specific cell called T-CELL are
also activated. This cells interact with the antigen in
several ways collectively known as CELLULAR
• For an infection to be established, the microbs
must overcome many substance barrier: Skin,
degradative enzymes and mucus.
• Any orgs.
that penetrate these barriers
encounters two levels of resistance:
• (i) Non-specific and (ii) specific immune
• Immunity – General ability of a host to resist a
particular infection or disease.
• Immunology – Is the science that is concerned
with immune response to foreign challenge and
how these responses are used to resist infection.
• There are two fundamentally different types of
immune response to an invading organisms.
• Non-specific and innate/natural immunity.
• Generally, animals (man) have a number of
resistant factor which defend the body against
invasion by pathogenic organism.
• The non-spectacular host defense system
does not involve immune response and they
are not induced by the presence of foreign
molecules, they are natural host defense
• They include (i) Anatomical defense (ii) diet
(iii) Age (iv) Stress.
• (i) The anatomical defense system: are natural defense
system of the body.
• The system include the skin and the linning of the
openings of the body to the environment. The skin is
an effective barrier to the penetration of microbes.
• However, microorganisms can get into the body
through the skin when its damaged.
• The entry of microbes through the nose or mouth are
protected by the action of ciliated epithelial cells and
the mucus substances of the nose and throats.
• The acid nature of the stomach (pH 2) kills potential
pathogens through oral means.
• Those that are resistant to this pH may be eliminated in the
small and large intestine that have higher pH value.
• In addition, competition of such pathogen with large
number of resident organs of the digestive system help
to eliminate them.
• Since they are fewer in number, the kidney and the
substance of eye are both secretion containing enzymes
(lysosome) which reduce microbial population.
• Diet: play a role in host resistance, highly malnourished
individual is more prone to infection than the highly-fed.
• Protein shortage from diet may alter the composition of
the normal floral of the body thereby allowing
opportunistic pathogens a better chance to multiply
• Not eating a particular substance needed by a pathogen
may help to prevent diseases such as dental caries in the
teeth, depends on sugar on which it feeds.
• Age: The young and the aged are more liable
to infection than others.
• In the young, the establishment of nonpathogenic organs are less since maternal
immunity has disappeared.
• In the elderly, the immune system is
weakened as a result of series of exposures to
pathogenic organism and hence certain
diseases, this therefore and the establishment
of pathogen.
• Stress:
The immune response is highly specific (i.e.) the
interaction between antigen and antibody or
interactions between T-Cell and antigen is specific.
• In immune response each new pathogens must
react with immune system to produce a response
which consequently lead to the production of
antibody against the specific microorganism.
• This response may not occur until after several days
of contact but when it does occur it is limited against
the antigen microorganism
• Once the immune system has been stimulated to
produce specific antibody or activated T-Cells (i.e.)
stimulation of the body by the same antigen result in
rapid production of very large amount of the same
antibody or T-activated cells.
• It is this capacity for memory that allows the host to
resist subsequent interaction by the same pathogen.
• It is this principle that is used in vaccination for
protective immunity.
• This is a state whereby the immune response
bears to distinguish between non-self (foreign)
macromolecule of host (self
• Macromolecule and hence does not destroy its
own cell.
• This is very important because the
macromolecules of the host are also potential
• This host molecule will be damaged or destroyed.
If the host produce specific antibody that
recognize them as foreign antigen
• Is a molecule that is synthesized by an animal in
response to the presence of foreign substance
known as antigen.
• Each antibody is a protein molecule called
• ANTIGEN: Is a molecule which can cause antibody
• All cells possess antigen in their cell surface
membrane which act as makers that enables cell to
recognize each other.
• Antigens are usually protein or glycoprotein that is
protein with CHOtail.
• The body can distinguish its own antigen (self) from
foreign (non self) and normally only makes
antibodies against non-self antigen.
• The immune system involve a number of cell
type and organs that interact in various ways
to bring about immune response.
• The organs of the immune system include:
• Thymes
• Spleen
• Bone-marrow
• Lymph nodes
• The most important cell involve in immune
responses is the lymphocyte.
• Lymphocytes are type of white Blood cells
that are disposed through out the body in the
blood circulation.
They arise from
indifferientiate stem cells in the bone marrow.
• There are 2 types of Lymphocytes, these are:
• B – Lymphocytes
• T – Lymphocytes (T-Cells)
• The two (2) types of lymphocytes are derive
from the bone marrow but their
differentiation is determined by the organ by
which it become establish.
• The B – Lymphocyte mature in the Bone Marrow
while T – Lymphocytes mature in the Thymes.
After maturation, the B and T – cells are disposed
throughout the body via the blood circulation.
• They may then reside in the spleen or lymph
• One major difference between the B-cell and Tcells is that the B – cells are responsible for
antigen reaction and Anti-body production while
the T-Cell have specific antigen including site on
their cells surfaces, thereby they interact
specifically with the antigen.
• There are different type of the T-cells, these
• T- Helper Cells (TH) stimulate B Lymphocytes to
produce antibodies.
• T- delayed hypersensitivity cells (T2) are involved
in hypersensitivity usually by activating macrophotosynthesis.
• T cytotoxic cells (T2) destroy pathogen/antigen by
lysing them
• T Suppressor cells(TS) regulate immune response.
• Other cells that are involve in immune responds
that consequently lead to the protection of the
body against infections include
• Antibodies
• Macrophages
• Natural Killers of cells (NK)
• This are also known as immunoglobins. This are
protein molecules that are capable of combining with
antigenic materials on the surfaces of antigen that
stimulate there production.
• They are found in the serum function of the Blood
and in some other body fluids.
• There are 5 major classes of immunoglobins and these
are classified as:
• IgG, IgA, IgM, IgE, IgD
• IgM is the first immunoglobin to appear following
• In most individuals about 80% of all immunoglobin is
IgG which means it is the most circulating antibodies in
the blood.
• This are large phagocytic Cell which are capable
of ingesting antigen and destroy them.
• They are abundant in spleen and can engulf
particles as large as Bacterial cells.
• They kill Pathogen/antigen by releasing lytic
substance particularly protease, lysosyme and
• They are also involve in the early step in antibody
synthesis. Here they serve to poses cells for the
antibody products.
• The T-cells which are also involved in immune
response have different function for example:
• TH - Stimulate B – Lymphocytes to produce antibodies
• TD – Are involve in Hypersensitivity reaction, usually by
activating macrophages
• TC – Destroy antigen or pathogen by lysing the cells
• TS – Regulate immune response
• (NK) Natural Killer Cells
• These are class of lymphocytes that play a role in
destroying foreign cells. They are produce in the
absence of Antibody
• They are capable of destroying malignant cells and
viruses infected cells without previous expose to the
• The action result are thought to be the release
chemical substance which are toxic to the Antigen.
(They destroy cells by secreting cytotoxins)
• They are modulators of immune response release
by T-Cells, Macrophage cells of immune system.
• They include: interleukins, interferon, chromatic
factor. Macrophage activation factor. Migration
Inhibition factor.
• Interleukins – Cause proliferation of B –
lymphocytes and enhance antibody formation.
• Interferon – Coat other cell surface to prevent
• CF – Attract macrophage to site of antigen.
• MAF – stimulate macrophage to become more
effective killer
• MIF – prevent macrophage away from the
• The term antibody (immunoglobulin) refers to a group of related globula
protein which are capable of specific non – covalent binding to the molecules
which induces their formation.
• They can also be defined as Globula Protein Produce in response to an
introduction of antigen to foreign body.
They are found in the serum fraction of the blood and in some other body
fluids. They are 5 major classes of Antibodies/Immunoglobulin designated as:
• However, all the antibodies have certain
structural features in common.
• The basic unit of each antibody molecule is a
complex of four (4) Polypeptide chain.
• It is composed of 2 identical heavy chains
(longer) and 2 light chains (short).
• The 2 light chains are identical in Amino acid
sequence (i.e. arrangement of amino acid in
polypeptide chains).
• The long and short chains are linked together
by disulphide bonds.
• When an antibody is treated with an enzyme
such as papain, it’s broken into several fragment.
• The 2 fragment that contained both the light
chain and half of the long chain is called Fab
• The Fab fragment are the portion that combine
the antigen and therefore determine antigens
specificity of the body.
• The precise portion of the Fab fragment into
which the antigen fixed is known as the
• The remaining fragment of the anti body
molecule, which is composed of half of the long
chains is refer to as the FC fragment.
• The FC fragment does not bind the antigen but
determines the bindery of an antibody to
specific host cells.
• The antibody found in human and other
vertebrates have some structural variations.
• For example, IgG has a structure similar to the
monomeric unit of all antibody.
• On the other hand, IgA is dimeric (2 unit)
• The 2 units are held together by disulphide
• The production of antibody in response to stimulation by
antigen is a complex process. However, it involves
interaction between the cell surface of T-cells, B – Cells
and Antigen presenting cells (APC).
• The APC” could be macrophages or B-cells when an
antigen is introduce into the body. The antigen must be
presented by APC”.
• In these sequence of reaction, the APC has antigen
receptor on the cell surface. The antigen binds the
receptor site.
• Consequently, antigen is engulf by a process known as
ENDOCYTOSIS. The antigen is then digested or processed
and then present the complex to TH
• (T-helper cells).
• The TH cells then stimulate the B-cells through the release
of chemical substances known as INTERLEUKINS
• Consequently, the B-Cells divide to produce
several copies of it-self. The B – cells then
differentiate to form large antibody secreting
cells known as the PLASMA cells.
• In addition, special B – Cells known as the
MEMORY CELLS are also produced.
• The plasma cells lives within one week during
which it secrete Antibody.
• The memory cell on the other hand remain in
circulation for several months, when the body is
re-exposed to the initial antigen, the memory
cells are transformed to plasma cells which then
secrete large quantity of Antibody.
• An antigen is a substance that when introduced into an animal
body reduce immune response which may lead to antibody
production or the production of activated T-cells.
• The antigen is also capable of reacting with either the
antibody or TC receptor.
• One other major property of an antigen is that it must be of
fairly high molecular weight usually 10,000 and above. There
are 2 major classes of an antigen these are:
• Soluble Antigen
• Insoluble Antigen
• Soluble antigen are/includes Proteins, Toxins, Polysaccharide,
lipids and Nucleic acid.
• Insoluble antigen includes cells of bacteria, fungi, spore and
propagules of fungi.
• Usually, the entire portion of an antigen may not be antigenic
but only a distinct portion of the molecules, that position on
the antigen molecules is called its ANTIGENIC DETERMINANT
• In the body of vertebrates (e.g.) Man stimulation of
immune responds due to several antigenic
determinants may lead to their production of a
group of antibody.
• Such group of antibodies are referred to as
• When a single antibody is produced as a response to
a single antigenic determinants such is referred to as
• These are group of antigen (EXOTOXINS) that have a
completely different mechanism of action from other
type of toxin or Antigen.
• They have the ability of binding Tc cells at a
site outside the normal antigen binding site.
The binding may stimulate the production of
many Tc cells.
• The massive stimulation of the TC cells result
into cell mediated response which may result
into generalize shock known as TOXIC SHOCK
• Example includes the Staphylococci. Shock
syndrome Toxin.
• The Staphylococcus enterotoxin produced by
Staph. aureus are also example of super
• The study of antigen antibody reaction in-vitro is
SEROLOGICAL because it involves reaction that use preparation of
antigen and antisera (Serum containing antibody).
• This study is very important in clinical diagnostic microbiology. A
number of serological reaction can be observed depending on the
Nature of the antigen, the antibody and the condition of reaction.
• Generally the principle of antigen antibody reaction is based on he
specific combination of antigenic determinant with a variable
region of the antibody molecules resulting into final variable or
measurable results.
• This combination serve to disturb the antigen sufficiently to either
reduce or totally eliminate its biological activity.
• Example of typical antigen antibody reaction includes precipitation,
agglutination, Lysis, killing, immolizes, neutralization, phagocytoxins
• Some of these reaction have been widely used in the diagnosis of
the disease for pregnancy test and urine test, for drug abuse such
as connabanoids (marijuana), cocaine, opiates steroids etc.
• It is a reaction between antibody and soluble antigen which
result into visible mass of antibody antigen complex in form of a
precipitate. Precipitation reaction can be carried out in a Test
Tube or on dgar gels (immunodiffussion).
• Some tube containing fixed concentration of antisera (antibody)
are set up. Different concentration of antigen are prepared an
equal volume of each concentration introduced into one of the
• The mixture is slightly shaken and incubated for some minutes.
Precipitation is then observed in each tube. Precipitation
occurs maximally any where there are optimal proportion of the
antigen and antibody.
• When either reactions occur at excess. The formation of large
antibody aggregate is not possible.
• Antigen antibody reaction on agar gel. This
technique is very useful on the study of specificity
of antigen antibody reaction.
• Immunodiffussion techniques: wells are cut in
the agar gel and such wells are separately filled
with the antigen and the antibody.
• Both the antigen and antibody diffuse from the
separate wells and precipitation bands form in
the region where the antigen and Antibody meet
on optional proportion.
• The shape of the precipitation bands is an
indication of the relatedness of the antigen. If
two antigens in adjacent wells are identical, they
will form a fused precipitation bands referred to
as a line of identity.
• Immunodifusion is often used as a tool in
biochemical research to access the relatedness of
protein from different sources. Immunediffusion
by (Ring test) carried out in test tubes.
• Method: Tubes containing Molten Agar into
which different concentration of antiserum has
been incorporated are placed in test-tube.
• Agar is then laid with equal volume of antigen.
• Incubate and watch for precipitation bonds at
point of optimal proportion.
reaction have also been used to diagnose
diseases elicited by soluble toxin.
• It is a reaction between antibody and an insoluble
antigen which result into a visible clous of particles.
The procedure can be carried out in a test-tube or glass
slide. In a test-tube different concentration of antisera
are put in test-tube. Add suspension of the antigen,
incubate for minutes and observe for agglutination.
• Serum is placed on the microscopic slide is then mixed
with the antigen incubation is done for a short period,
observe under the microscope for clumping.
• This reaction form the basis for the classification of blood called
• The principle of agglutination test have also been used for the
identification of pathogenic streptococci Neaserea – gonorrhea,
Candida allaican, Homophiles influenza, Hepes simplest Virus aid in
the diagnosis of diseases one.
• Neutralization of microbial toxins can occur when the antibody
molecule directed specifically against the toxin, combine with the
toxin at its active position that is responsible for cell damage is
• An antiserum containing an antibody against the toxin is referred to
as antitoxin.
• The principle of this reaction is being used for the diagnosis of
diseases coming from toxin elicited by micro organism e.g.
Clotridum botulium
botulism and
Clostridum tetanus Alpha toxin produce by Staphylococcus aureus cell
that cause pyrogene infection.
• Clostridium perfirenges (caused food poisoning) it usually lyse
hormone in action.
•Complement System:
•Antigen – Antibody and cell medicated characteristics are
important in immunity.
•The consequence of this characteristics is brought about by
either precipitation, agglutination or neutralization of either the
antigen or its product.
• However, there are several other kinds of antigen antibody
characteristics that would not otherwise occur except in the
presence of other group of serum substance called
•Compliment is a series of enzyme found in serum that has
contact with specific antibody to bring about several kind of
antigen antibody characteristics.
• The major feature of complement system are:
• System is a series of enzyme in the body
• The enzyme of a system are normally inactive
but become active or activated when an
antigen antibody reaction occur.
• The enzymes attack bacteria cell or other
foreign cell causing lysis or leakage of cellular
consequent as a result of damage of the cell
• They act on the antigen only when an
antibody to that antigen is present.
• Some characteristics in which complement participate
• Lysis of Bacteria cell particularly the G-ve
• Microbial killing by all damage and leakage of the cell
• Phagocytosis: Especially in the infection caused by
microorganisms that possess a capsule.
• Another immunological characteristics involving the
compliment system include certain aspect of
inflammatory respond and the production of protein
• Anaphlatoxin causes the release of Histamine when
they banding most cell.
Histamine include
inflammation and made the capillary most permeable
thereby leading to leakage of leukocytes and fluid to
escape into the tissue cell, this may result in
Anaphylatic shock.
• In Complement Fixing System, the bacterial
suspension (or other antigen) and the
complement are mixed.
• If the antibody against the antigen is present
in the serum, the compliment is fixed.
• In this system haemolytic antibody (hymolysin) is prepared by
immunizing rabbits with the red blood cells of sheep.
• This indicator system is then added to the complement fixing
• If complement had been fixed, by being used in the reaction
between the test antibody and antigen, no haemolysis of the
Red blood cells of sheep.
• This indicator system is then added to the complement fixing
system. If compliment had been fixed, by being used in the
reaction between the test antibody and antigen, no
haemolysis of the Red blood cell will occur since the
compliment has been fixed.
• This is a positive (+ve) complement fixation test indicating the
presence of a specific antibody to the antigen.
• In case of Negative (-ve) complement fixation test haemolysis occur
because the important reaction of a (+ve) tuberculin test is a tissue
hardening that can easily be fill about 10mm or more in diameter
occurring in 48 – 72 hours, following intra dermal injection. This
indicate immunity.
• All person who have had and overcome tuberculosis infection
(Natural or resulting from vaccinator) generally have some degree
of immunity to further infection.
• A number of skin test to show past or present infection with
Bacteria, fungi or virus includes:
• Tuberculin
caused by
• M. tubercleae
• Lepromin
caused by
M. Lepreae
• Brucellergin
caused by
Brucella sp
• Mumps
caused by
Virus, paramyxovirus
• Complement is free to be fixed by the Red blood cell (antigen) and
the antisheep RBC (hymolyson antibody.
• Summarized diagrammatically
• The complement fixation test is useful when the test antigen and antibody
combination does not give a visible reaction such as agglutination and
• It is used in the laboratory to diagnose many infectious diseases including
those of Bacteria, Viral, protozan and fungal antigen.
• One of the best known application of the test is the Wesserman test for
• .
• This is a serological diagnostic test for syphilis. The reaction detects
Wesserman antibody in the serum of syphilis patients.
• The test is carried out by reacting the serum of patient with Cardiolipin –
Lecithin - Cholestrol (CLC) antigen which undergo complement fixing
If the serum contains antibodies for Treponema pallidum, the antibody is
fixed with the antigen (CLC). Hence where exposed to the indicator sheep
RBC, <no lysis occurs – a positive> reaction. <In –ve reaction lysis of the
RBC will occur>.
• Introduction:
• Hypersensitivity is an immune over reaction caused by
either antigen – antibody reaction or cellular immune
processes but usually harmful to the animal.
• There are 2 main types viz
• Immediate type Hypersensitivity
• Delayed type Hypersensitivity
• This is an antigen – antibody mediated immune reaction.
• Immediate type hypersensitivity commonly involve allergies
and depending on individual .
• The type of antigen may cause mild or extremely severe
generalized reaction which can lead to death if not treated
• Antigen that cause these hypersensitiveness are
known as Allergens.
• Example include: Pollen and fungal spores,
Insect Venoms (by sting), Penicillin and some
other drugs, certain food etc.
• Typical symptoms of allergic reaction caused by
immediate type hypersensitivity are sneezing,
wheezing, running nose, tears in the eyes, itching,
and development of rashes.
• In all these hypersensitivity reactions, the first
contact with the allergen cause the production of
IgE instead of circulating like other classes of
antibodies such as IgG or IgM.
• .
• The IgE molecules tend to bind or get attached
to the surface of the mast cells and Basophils
White blood cells and leukocytes).
• Subsequent exposure to the allergies cause
the IgE molecules attached to these cells
(mast, Basophils) causing the bridging of at
least 2 IgE molecules.
• The release of chemical mediators such as
Histamine and Serotonin (modified aa) or
small peptodes from the mast cells and
• The release of these chemical mediators
causes dilation of blood vessels and
contraction of smooth muscles resulting in the
typical symptoms of immediate type allergies
• Reaction is usually of short duration but an
individual can respond repeatedly by
subsequent exposure to the allergen.
• These reaction are generally referred as
• The magnitude of the anaphylactic reaction
could be mild or to
• such severe symptoms that the individual goes
into Anaphylatic shock.
• Anaphylactic shock is characterized by severe
respiratory distress, sharp drop in blood
pressure due to capillary dilation (leading to
extrusion of leukocyte and fluids into the
tissue cell flushing) and itching.
• If not treated, immediately with large doses of
adrenalin to counter smooth muscle
contraction and promote breathing, death can
• Delayed type – hypersensitivity is an overreaction of the cell mediated
immune response. The reactions involves the activities of a subset of Tcell called T-delayed – Type hypersensitivity (TD) cells. Delayed type –
hypersensitivity appear only after 18 hours. Persistent for some days and
are not suppressed by anti histamines.
• Delayed type hypersensitivity reaction occur against invasion by some
micro-organism (e.g.) M. tuberculosis, skin contact with some chemicals
(e.g.) cosmetics and certain chemicals (content dermatitis).
• On exposure of the skin to the agents, the skin feels itchy at the site of
content which within several hours redding and swelling appear. This is an
indicative of inflammatory response. The TD cells then secrete
lymphokines which then attract white blood cells and macrophages to the
site of content. Localized tissue distruction then occur due to the lytic and
phagocytic activities of these cells.
• The characteristics skin reaction seen at the
site of injection is a result of an inflammatory
response arising as a result of the release of
lymphokine by activated T-Cells.
• The tuberculin test is a typical delayed type of
hypersensitivity test. It is performed with
purified protein derivative (tuberculin).
Injected subcutenously into the skin.
• They
microorganisms which are capable of inducing
host damage.
• There are different types of toxins but can
generally be classified into 3 groups.
• These are toxins produced and released
extracellularly as the microorganism grows, most
often they travelled from the point of injections
to distance part of the body where they cause
damage to cells, tissues and organs.
• Examples of exotoxins are:
• Tetanus toxins
• Diphtheria toxins and
• Botullinum toxin
• Generally, exotoxin are
• proteinous in nature and are excreted by certain
G+ve and
• G-ve bacteria.
• They are heat liable.
• Their mode of action are:
• Specific with defined specific actions on cells
or tissues.
• They are highly immunogenic,
• They can be destroyed with formaldehyde or
formalin but still retain their immunogenic
• Generally they don’t produce fever in host
• They are exotoxins that act on the small
into the intestinal lumen thereby leading to
symptoms of DIARRHEA.
• They are produce by various of G+ve bacteria
including the food poisoning organism example of
organism that produce enterotoxin include S.
aureus, C – perfrigenes, B. Cereus
• Enterotoxins have similar properties as the
• The major difference is that enterotoxins are
produced mainly in the intestine and do not
travel to distance part to effect their actions.
• They are toxins produced by certain G-ve
bacteria which remain bond to the cell but
release in large amount only when the cells lyse.
• In most cases endotoxins are lipopoly
saccharides which are part of the outer layer of
the cell wall.
• They are released from the cell only when the
cell lysis
• Generally on their mode of action.
• They include fever, diarrhea and vomiting in
other words they are not as specific in their
mode of action as an Exotoxins.
• They are heat stable.
• They are weakly toxic (i.e) large amount are
required to exact their effect.
• They are relatively poor immunogenic.
• They can not be used to produce toxoids.
• Example of organism that produce endotoxins
include E. coli, Salmonella, Shigella.
• This assay technique have been developed for
the determination of the presence and possibly
quantity of endotoxins in fluids.
• It is carried out by using suspension of
Amoebocytes cells from the horse shoe crab
called Limulus polyphemus.
• Endotoxin cause degranulation and lyses of the
cells. The degree of characteristics is measured
• To carry out this test, a suspension of
Amoebocytes cell are prepared in tubes and the
fluid containing the endotoxins is added.
• After incubation at 37o C for 1 hr. The degree of
turbidity is determined by using spectrophotometer. If
cells are lysed by the endotoxin, the turbidity of the
suspension decreases.
• This method is highly sensitive and can be used to
determine quantity of endotoxin as low as 10
picogrmm per ml.
• This assay method has been used to detect the
presence of minute quantity of endotoxins in serum,
C.S.F., drinking water and fluids use for injections.
• Generally, toxicity of toxins is measured in terms of the
amount of toxin that all kill 50% of population of
experimental animals.
• This is often referred to as LD50.
• A substance produced by the body cells as a
reaction to toxin produced by bacteria which
neutralizes their action.
• An antiserum containing an antibody that
specifically neutralize a toxin is called
• This Neutralization reaction occurs when the
toxins molecules and the antibody molecules
directed against the toxins combine in such a
way that the active portion responsible for cell
damage is blocked.
• Is a term used for toxin which have been modify so
that it is no longer toxic but it is still able to induce
antibody formation.
• One of the common ways of converting a toxin to a
toxoid is by treating it with formaldehyde which
blocks some of the amino groups of the toxin.
• Thus the toxin is render inactive but still capable of
inducing antibody formation. Some examples of
• In the production of toxoid, the organism is first
grown under a condition which it will produce toxins.
• For example in the production of diphtheria, toxin is
produced by Corynebacterium diphtheria grow
under Anaerobic condition on a medium consisting
essentially of hydrolysis of protein from horse meat
under the condition of limiting Fe2+.
• The organism produce diphtheria toxin at the end of
culture period, cells are removed by centrifugation.
• The filtrate is then passed through bacteriological
membrane to remove any cell debris.
• The filtrate thus obtained is incubated with formalin
at 37oC.
During this incubation the toxin is
inactivated and its activity is constantly checked by
ingestion into experimental animals.
• They are substances which when introduced into the body
provide immunity against infection.
• There are 3 major type of vaccines, these are:
• The live attenuated organism
• Killed vaccines
• Toxoids
• Live attenuated Organism: Consist of living pathogens where
virulence has been reduced by passing them through host
different from the usual host.
• Alternatively, the non-virulent stains of the pathogens may
be used in both cases.
• The organism are still antigenic in other words they are still
capable of inducing antitoxic body formation but can no
longer cause infections.
• Examples
of live vaccines include those against Polio,
Mumps, Measles, Rabbies and Yellow fever vaccines.
• Killed Vaccines: This consist of suspension of
fully virulent organism that has been killed midly
in other not to destroy the antigen.
• The killing can be done or be achieved by
heat.(60oC for 1hr).
• Killing can also be done by the use of chemicals
such as formalin, Alcohol and Phenol, in some
cases, UV-radiation may be used for the mild
• Example of killed vaccine include those against
whooping cough, cholera, poliomylites and those
against Typhoid fever.
• Toxoids – refer to earlier Note.
• Viruses multiply only in living cells.
• Viruses to be used for vaccine production therefore
must be grown in such cells. Usually such viruses are
cultured when introduced into tissue culture.
• Tissue or cell culture are growth of animal cells invitro in
monolayers. Examples of such is the tissue culture on
the Kidney of a monkey.
• This has been widely used in the production of polio
• In the production of Polio vaccines, cells of the kidney of
Rhesus monkey are used for the production of the
tissue culture on which the virus is grown.
• In this process the kidney is first macerated and treated
with trypsin in other to separate individual cells.
• The suspension by cell is then distributed in shallow
container (e.g.) Glass plates and covered with suitable
• After this, a cell is incubated at 37oC for 4 – 6 days.
During incubation, a confluent of growth of a
monolayer of cells is obtained at the end of the
• The culture fluid is removed and replaced with
maintenance medium (Not containing any protein).
Live viruses are then inoculated into the tissue culture
and incubated at 37oC form 4 days.
• The virus particle are then arrested by Centrifugation.
• The suspension of viral particles obtained are then
inactivated with formalin.
• The success of inactivation is checked by injection into
Embroyonated egg or experimental mammals.
• The most important activity of the microbiologist in
medicine is to isolate, characterize and identify causal
agents of infectious diseases with a view to advising the
physician in matter relating to the diagnosis and
treatment of such diseases.
• In the past the clinical microbiologist is able to carry out
this task within 48 hours of sampling.
• Today, diagnostic methods based on immunology have
drastically reduce this time to the extent that it’s
possible to positively identify pathogens in minutes or
• This techniques are based on the principle of antigen –
antibody reactions.
Such as Agglutination,
Neutralization, precipitation, complement fixation etc.
• In addition to these diagnostic procedure.
Immunological reaction have also been
used in screening of urine for banned
drugs abuse (e.g) screening of athletes for
steroids etc and also in pregnancy test,
blood typing etc.
• The ease of use of these techniques in
diagnosis have been made possible for
packaging into kits which are most
effective and easy to handle.
• The kits also provide for mass screening
• The ease possible by now that community available
antisera prepared against several different antigen
from known pathogenic microbes are readily
available for chemical use.
• Positive to coat innert object such as latex, beads,
paper strips, microtilus with for the ease of use.
For the purpose of this course, we shall discuss
some of these techniques and their application viz:
• Agglutination Test
• Immunoblet (Immunodiffusion)
• RIA (Radioimmunology)

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