Introduction to Immunology

Immunology:

The science of studying immunity or studying the defense mechanisms

developed by the body against foreign pathogen.

What is immunity??

 It is derived from Latin word “immunitas”: exemption from civic

duties and prosecution “military service or paying taxes”.
 It means protection from disease and especially infectious diseases.

Immune system:

 Cells and molecules involved in protection of the body.

Immune response:

 The response to introduction of foreign agent.

 NOTE: Not all immune responses protect from disease; some
foreign agents, such as allergens, cause disease as a consequence
of inducing an immune response.

Autoimmune diseases:

 Some individuals mount immune responses to their own tissues as

if they were foreign agents.
 Autoimmune diseases like: diabetes and rheumatoid arthritis.
 Most individuals do not suffer from autoimmune diseases because
they developed tolerance towards their own (self) tissues.

*Immunity

1-Innate 2-Adaptive
1-innate immunity:

 Nonspecific.
 Natural immunity
 Refers to the ability of the host to resist microbial infection without
prior exposure.
 It does not require the generation of specific molecules and cells
 Rapid.
 It includes:
1. Skin and mucous membranes
2. Secretions (excluding immunoglobulins)
3. Serum factors (complement and acute phase proteins)
4. Phagocytic cells.

2- Adaptive immunity:

 Microorganisms or molecules which evade or overwhelm the

innate immune systems may interact with the immune system and
stimulate an adaptive response.
 Adaptive response may be: specific or nonspecific.
 Nonspecific adaptive responses include:
1. Increased tissue proliferation
2. Induced expression of antimicrobial peptides and proteins by
‘nonmilitary personnel’ such as epithelium
3. Increase phagocyte activity.
 Specific immunity is an interaction between ‘big things’ and their
receptors; i.e., B- cell or T-cell antigen receptors (BCR or TCR).

Anatomy of immune system

 Immune system is a complex of..

1- organs,
2- highly specialized cells and
3- even a circulatory system separate from blood vessels
 ……all of which work together to clear infection from the
body.
 Organs are called lymphoid organs.
 Function: it protect an individual from both outside invaders
and its own altered internal cells.

1-organs

primary lymphoid organs secondary lymphoid organs

*bone marrow *lymph node

*thymus *spleen

not essential for the

they are sites of development and generation of lymphocytes
maturation of lymphocytes but have a key role in the
maturation and
differentiation of these cells

Antigens and antibodies

 Antigens or immunogen:
 It is a substance that promotes the generation of antibodies and
can cause an immune response.
 It is a substance or organism recognized by the body as being
foreign and stimulates immune response.
 It has one or more antigenic determinant sites ‘epitopes’
 Epitope: the basic recognition unit “antigenic determinants”
(The smallest identifiable part of antigen that is bound by a
receptor)
  Immunogenicity: the ability of substance (immunogen) to
induce a specific immune response
 Antigenicity: the ability to induce a response and the ability to
react with the products of that response.
 Hapten:
A substance that is non-immunogenic but which can react with
the products of a specific immune response.
 Haptens are small molecules which could never induce an
immune response when administered by themselves but which
can when coupled to a carrier molecule.
 Free haptens, however, can react with products of the immune
response after such products have been elicited.

CHEMICAL NATURE OF IMMUNOGENS

 Proteins
the vast majority of immunogens are proteins. These may be
pure proteins or they may be glycoproteins or lipoproteins. In
general, proteins are usually very good immunogens.

 Polysaccharides
Pure polysaccharides and lipopolysaccharides are good
immunogens.

 Nucleic Acids

Nucleic acids are usually poorly immunogenic. However, they may

become immunogenic when single stranded or when complexed
with proteins.

 Lipids
In general lipids are non-immunogenic, although they may be
haptens

 Antibodies:
 Antibody (Ab):
A specific protein which is produced in response to an
 immunogen and which reacts with an antigen. Or they are
members of a group of soluble proteins collectively known as
immunoglobulins (Igs).

 Found in serum, which is the fluid portion of coagulated

(clotted) blood, tissue fluids and some secretions.
 Abs are proteins that are made in response to Ag and can
recognize and bind to that antigen.
 Highly specific.
 Each antibody has at least 2 identical sites that bind to antigenic
determinants. These sites are known as antigen- binding sites
‘paratopes’
 Paratope: the area of antibody molecule that interact with
epitope
 No. of Ag-binding sites is called the valence of that antibody.

 Structure of Antibody:
 Consisting of 4 polypeptide chains …
-Two identical large or heavy (H) chains
-Two small or light (L) chains
Chains are linked by disulfide bridges and other bonds to form a Y-
shaped molecule.
 There are 5 immunoglobulin classes or isotopes
(IgM, IgA, IgG, IgE& IgD)

 Functions of antibodies:
 Recognize antigens
 Mark them for destruction
 Coat the foreign invaders to make them attractive to the circulating
scavenger cells, phagocytes, which engulf microbe (opsonization).
 When some antibodies combine with Ag, they activate a cascade of
nine proteins, known as complement, that have been circulating in
inactive form in the blood. It forms a partnership with antibodies,
once they reacted with antigen, it helps destroy foreign invaders
and remove them from the body.
 Other types of antibodies block viruses from entering the cells.
 Differential leukocyte counts of blood

Principle:
 The differentiation of leukocytes is based on the size and shape of
nucleus and cytoplasmic granules staining.
 Out of several diagnostic tests, differential leukocyte counts of blood
are the most common. The test provides valuable information about
the differential diagnosis of bacterial and viral infections as well as
anemia or leukemia.
 Neutrophil count is usually higher with some exceptions during
bacterial infection and monocyte counts in viral infections.
 The normal leukocytes blood profile contains:
50-70% neutrophils, 20-30% Iymphocytcs, 2-6% monocyte, 1-3%
eosinophils and about 1% basophils.
 A blood smear is stained either with Wright's stain or leishman
stain.
 These stains differentiate the granuulocytes (polymorphonuclear
leukocytes. basophils and eosinophils). agranulocytes
(mononuclear leukocytes) and thrombocytes (platelets).
 Table: the abnormal cases of (WBCs):

Cell Increase due to Decrease due to

Neutrophil 40-60% Acute infection. Viral and bacterial

infection.
(3- 5 lobes) Acute stress.
Influenza.
Gout or thyroiditis.
Chemotherapy.
Myelocytic leukemia.
Radiation therapy or
exposure.

Lymphocyte 20-40% Chronic bacterial Chemotherapy.

infection.
(rounded nucleus) HIV infection.
Hepatitis.
Steroid usage.
Lymphocytic leukemia.

Monocyte 2-8% Chronic inflammatory Chemotherapy.

diseases.
(kidney-shaped Steroid usage.
nucleus) Parasitic infection.

Tuberculosis.

Eosinophil 1-4% Allergic reaction. Chemotherapy.

(head phone- shaped Cancer. Radiation therapy.

nucleus)
Collagen vascular
disease.

Basophile 1% Allergic reaction. Cancer.

(S- shaped) Varicella infection. Sever injury.

Chronic myelogenous Acute infection.

leukemia.

REMEMBER: Never Let Monkey Eat Bananas

Requirements
a. 70% ethyl alcohol
b. Sterile lancet for pricking or syring
c. Container with disinfectant
d. Leishman stain or Giemsa stain

Differential count steps:

1-Clean a slid from dust and wax very well.

2- Put one small drop of blood at one end of slide.

3-Take another slide and place it along the drop of blood and allow the
blood to spread to edges of the spreading slide. Hold the spreading slide
slightly slanting position (45-50◦) and push the spreading slide other end
of blood drop slide rapidly to form a thin smear with no ridges. Leave the
smear to dry before staining.

4-Cover the smear with leishman's stain for seconds and add drops of
distilled Water then let it stain for 5 min.

5-wash with distilled water and leave to dry completely then examine
under microscope at 40X or oil lense.
6-Count at least 100 white blood cells and make a record of each type of
leukocytes seen.

7-Calculate the percentage of each cell types recorded by you.

Percentage = Number of specific cell type /total number of cells

X100

Record your result:

Cell Basophile Eosinophil Monocyte Lymphocyte Neutrophil

Number

Note:

Leishman’s stain contains:

1-The methanolic stock solution that directly fix the smear eliminating a
prefixing step.

2-1st methylene blue dye, a basic dye, which gives color to an acidic
component ”amines” (basophils, neutrophils).

3-2nd eosin dye, an acidic dye, which gives color to a basic protein
component (eosinophil, neutrophil)

 These dye differentiate the different component of blood.

 It uses to stain leukocytes, malaria parasite and trypanosomes.
 Immunization

Immunization, or immunisation,
 Is the process by which an individual's immune system becomes
fortified against an agent (known as the immunogen).
 When this system is exposed to molecules that are foreign to the
body, called non-self, it will orchestrate‫ينسق‬an immune response,
and it will also develop the ability to quickly respond to a
subsequent encounter because of immunological memory. This is a
function of the adaptive immune system. Therefore, by exposing an
animal to an immunogen in a controlled way, its body can learn to
protect itself: this is called active immunization.
 Immunization is done through various techniques, most
commonly vaccination. Vaccines against microorganisms that
cause diseases can prepare the body's immune system, thus helping
to fight or prevent an infection.

 Immunization can be achieved in two manners:

immunization

active passive
immunization immunization

Active immunization
 The introduction of a foreign molecule into the body, which causes
the body itself to generate immunity against the target. This
immunity comes from the T cells and the B cells with their
antibodies.
  Active immunization can occur naturally when a person comes in
contact with, for example, a microbe. If the person has not yet
come into contact with the microbe and has no pre-made antibodies
for defence, as in passive immunization, the person becomes
immunized. The immune system will eventually create antibodies
and other defences against the microbe. The next time, the immune
response against this microbe can be very efficient; this is the case
in many of the childhood infections that a person only contracts
once, but then is immune.

Passive immunization
 Passive immunization is where pre-synthesized elements of the
immune system are transferred to a person so that the body does
not need to produce these elements itself.
 Currently, antibodies can be used for passive immunization. This
method of immunization begins to work very quickly, but it is
short lasting, because the antibodies are naturally broken down,
and if there are no B cells to produce more antibodies, they will
disappear.
 Passive immunization “natural” occurs physiologically,
when antibodies are transferred from mother
to fetus during pregnancy, to protect the fetus before and shortly
after birth.
 Artificial passive immunization is normally administered
by injection and is used if there has been a recent outbreak of a
particular disease or as an emergency treatment for toxicity, as in
for tetanus.

Method of Immunization:
1- Choice of animal:
a. Rabbit:
 Pure strain and about 2-3 Kg weight.
 Should be about one year.
 Amount of antigen which induce immune response=
50- 100mg.
 Rabbit are used most often to produce immune serum
because:
  Easily housed and easily cared
 Easy to handle
 Inexpensive
 Yield adequate volume of high- tittered serum
b. Mice:
 Antigenic material is low= 2- 20 mg antigen injection
and 0.01-0.1 mg induce immune response.
 Disadvantage:-
- give only 1ml of blood.
c. Chicken:
 Easily housed
 0.05- 5mg/ ml antigen emulsified in freund’s
incomplete adjuvant
 1.5-2 kg weight
 Disadvantage: high temperature of chicken body may
denature the antibodies formed so high osmoregulant
used
d. Other animals “goats, sheep or horse”:
 High yield of antiserum
 expensive

2- routes of immunization:
1. intravenous injection :
 is more effective when injected in several small
doses every 2 or 3 doses rather than in one large
dose
2. intramuscular:
 the antigen emulsified in equal amount of Freund’s adjuvant
“immune potentiator” (85% paraffin oil + 15% mannide
monoleate) ===> incomplete form
The complete form, Freund's Complete Adjuvant is contains inactivated
and dried”killed” mycobacteria in addition to the incomplete adjuvant.
3. subctanous
4. intraperitoneal
 5. intradermal
 Administration routes affect the quality of immune responses

Route of immunization is chosen according to:

1-animal species
2-antigen type
3-schedule of experiment

3- Preparation of specific bacterial antigen:

From Whole cell “untreated”:
1-bacterial cell grew in broth N.A. medium
2- Incubate at 37◦ c for 48 hr
3-Centrifuge at 10000rpm
4-resuspend pellet in phosphate buffer saline
5-Centrifuge at 10000rpm, resuspend in pbs and store.

4.a. Immunization of mice:

1- Fill the syringe with antigen (Ag)

2- Get rid of air bubbles
3- Inject 0.1 ml intraperitoneal.
 Primary immunization means inject Ag once then sacrifice
7days later
 secondary immunization means inject Ag twice
1st injection at day zero
2nd injection at day 7-8
Sacrifice at day 14-15
 4- b. Immunization schedule of rabbit:
NO. Of injection Time of Dose Method of
injection injection
1st injection 0 time 0.5 ml antigen Intravenous
“marginal vein”
2nd After 7 days 0.5 ml Ag +0.5 ml Intramuscular
incomplete
adjuvant ”shake”
3rd After 14 days 0.5 ml Ag +0.5 ml Intramuscular
incomplete
adjuvant ”shake”
4th After 28 days 0.5 ml Ag +0.5 ml Intramuscular
incomplete
adjuvant ”shake”

5- Collection of antiserum:
1- Slough animal
2- Collect blood
3- Leave blood to clot for 1 hr at room temp.
4- Heat blood at 37◦ C for 30 min “help to shrink the clot and increase
the yield of serum.
5- Put at refrigerator overnight
6- Centrifuge at 2000 rpm for 30 min “ to remove debris”
7- Filtration by filter with pore size 0.2 µm “bacterial filter”
Storage of antiserum:
1-Add 0.025 % sodium azide act as antibacterial agent at 37◦ c
2-Add equal volume of 50% glycerol and store at 4◦ c
3- Put at -20◦ C” deep freezer” without any additions.
 Subcutaneous

Intraperitoneal Oral with gastric tube

Preparation and preservation of plasma and serum:

Principle:

 Bleeding of animal should be done prior to feeding for

preparation of clear plasma or serum that has low lipid content.
Blood is collected with or without anticoagulant.
 Anticoagulants for plasma preparation
-Substances prevent clotting of blood & don´t interfere with most
of chemical reactions & minimize hemolysis as:
-Lithium heparin is added at 10 - I 5 µl / ml blood.
-EDTA
-Sodium acetate
-Sodium citrate

 Serum:
  When whole blood is allowed to clot, the supernatant is
serum.
 Blood serum is blood plasma without clotting factors (i.e.
Whole blood minus both the cells and the clotting factors).
 Serum includes all proteins not used in blood
clotting(coagulation) and all the electrolytes, antibodies,
antigens, hormones, and any exogenous substances (e.g.,
drugs and microorganisms).

Storage of serum:
In small aliquots at -20˚C or alternatively at 4˚C, after adding
preservatives like sodium azide 0.1%, merthiolate 0.01 % or
hydroxyquinoline sulphate 0.00001 %.

 Prepare serum or plasma from sterile blood under aseptic. It may

be stored after filter sterilization, by freezing or lyophilization.

 Serum can be prepared from plasma

By clotting fibrin present in plasma by adding calcium chloride and
warming it to 37°C. Two ml of 10% anhydrous Cacl2, is added to 10
ml plasma containing sodium citrate, EDTA or oxalate as
anticoagulant respectively. Break the clot after 2-4h and centrifuge at
3.000g for 30 min at 4°C.

-Blood plasma:
 The straw- colored /pale-yellow liquid component of blood that
normally holds the blood cells in whole blood in suspension.
 It makes up about 55% of total blood volume.
 It is mostly water (93% by volume), and contains
dissolved proteins (i.e. albumins, globulins,
and fibrinogen), glucose, clotting factors, electrolytes (Na+, Ca2+,
Mg2+, HCO3- Cl- etc.), hormones and carbon dioxide (plasma being
the main medium for excretory product transportation).

Plasma also serves as the protein reserve of the human body. It
plays a vital role in intravascular osmotic effect that
keeps electrolyte in balance form and protects the body
from infection and other blood disorders.
 Blood plasma is prepared by spinning a tube of fresh
blood containing an anticoagulant in a centrifuge until the blood
 cells fall to the bottom of the tube. The blood plasma is then
poured or drawn off.

* Plasma differ from serum:

1-Collected from whole blood contain anticoagulant
2-Contain clotting factor

Procedure
Serum:
1- Draw the blood and allow it to clot at room temperature for 1-2 h by
keeping the tubes in slanting position.
2-Carefully separate the clot from the wall of the test tube by using either
an applicator stick or Pasteur pipette or thin metal spatula. Avoid
haemolysis as it leads to degradation of immunoglobulins by enzymes
3-Aspirate the fluid collected above the clot. Centrifuge it at 2000-3000 g
for 10 min. Aspirate the supernatant and collect it in another tube.

4-Serum can be stored in frozen condition for months. It is advisable to

add preservative in serum to avoid any microbial or fungal growth.
Repeated freezing and thawing destroys many components of serum.
Hence the serum must always be stored in small lots in deep freeze (-
80°C). It can be stored without any preservative in deep freeze. Serum
can also be lyophilized and stored as powder.

Plasma:
Similar procedure is employed for preparation of plasma

1- Blood-containing anticoagulant is centrifuged directly at 2000-3000 g

for 10 min.
2- Aspirate the supernatant and collect it in sterile test tube. The deposit
may be discarded or may be used for preparing red blood cell
suspension.

Store and preserve plasma using the preservative and conditions

applicable for serum.
Serology: study of serum.

Why serum is preferred than plasma in serological reactions?

 Serum is preferred in serological assays due to presence of clotting
factors in the plasma which mask Ag – Ab interaction.

Serology
Definition:

It is the branch of immunology that deals with the in vitro study of

interactions between antigen (Ag) and antibody (Ab) in serum or other
fluids.

Antigen (Antibody generator)

 Antigen is any substance, which when introduced into the animal

body stimulates the immune system to produce immune responses
(Antibodies) specific to the injected substance and not to unrelated
materials.
 The surface of the antigen contains molecules are called: Antigen
Determinant Sites (ADS) or epitopes.

Antibodies

 A group of structurally related glycoproteins that are produced by

B-cells and plasma cells following stimulation of the host immune
system by an Antigen.
 Antibodies can binds specifically to the antigen molecules, which
stimulate their formation.
 An antibody molecule has at least two antigen binding sites (ABS)
(paratope )
Antigen- antibody interaction

The interaction of an Ag with its specific Ab to form an antigen-antibody

complex is mediated by multiple non-covalent bonds between the ADS
(epitopes) on the Ag and the ABS (paratopes) on the Ab.

Prozone or zone phenomenon

A false negative response resulting from high antibody titer which

interferes with formation of antigen- antibody complexes, necessary to
visualize a positive result.
 TYPES OF SEROLOGICAL TESTS

 Serological tests used to detect antigen-antibody interactions can

be classified into groups:

1-Primary Binding Assays: direct detection of antigen-antibody binding

(ELISA).

2-Secondary Binding Assays: detection of antigen-antibody binding

through the secondary changes that follows the primary binding
(Agglutination, precipitation, immunodiffusion)

 Serological tests could also be classified into two types according

to what will be detected, the antigen or the antibody into:

[A]– Antibody tests: These tests are applied to detect and measure
antibodies present in serum using known antigens.

[B]– Antigen tests: These tests are applied to detect antigens in

specimens or isolates using known specific antibodies.

Uses of serological tests

1-Diagnosis of infectious diseases.

2-Identification of microbial isolates.

3-Evaluation of immune status of the host.

4-Diagnosis of pregnancy.

5-Diagnosis of autoimmune diseases.

6-Blood grouping and tissue typing

7-Cancer diagnosis and prognosis through immunological detection of

tumor markers.

The Agglutination test

 In this reaction the antigen is part of the surface of some particulate
material such as a red cell, a bacterium or perhaps an inorganic
particle (e.g. polystyrene latex), which has been coated with
antigen.
 Antibodies added to a suspension of these particles combine with
the surface antigens and link them together to form visible
aggregate or clumps.

Agglutination/Haemagglutination:

 When the antigen is an erythrocyte or carried on erythrocytes

the term heamagglutination is used.
  The general term agglutinin is used to describe antibodies
that agglutinate particulate antigens.

 Interaction of antibodies with particulate antigens is called

direct agglutination”.

 However, interaction of antibodies with soluble antigens

(e.g. viral Ag, polysaccharide Ag or a hapten) carried on
carrier particles, e.g., latex is called Indirect or passive
agglutination test”.

The phenomenon is used for:

1. Typhoid fever caused by Salmonella Typhi in serum specimen taken
from suspected enteric fever cases (widal test).

2. Blood grouping

3. Pregnancy diagnosis

4. Determination of virus in blood e.g. mycovirus causes influenza.

Blood grouping slide agglutination

ABO blood grouping system
Red blood cells (erythrocytes) have certain proteins on their surface,
called antigens. Also, your plasma contains antibodies which will attack
certain antigens if they are present. ABO and rhesus are both types of
antigens found on the surface of red blood cells. There are lots of other
types but these are the most important.

Rhesus types (Rh)

Most people are 'rhesus positive'. This means they have rhesus antigens
(D) on their red blood cells. But, about 3 in 20 people do not have rhesus
antibodies and are said to be 'rhesus negative'.

The blood groups of the person can be detected by using antisera A,

B and D.

Procedure:
• Clean your finger with alcohol.
• Stab fingers with lancet and places 3drops of blood on slide,
labeled A, B and D.
 • Add a drop of anti A, a drop of anti-B sera and a drop of anti-D to
the slide.
• Using applicator stick mix the blood with the antisera on slide. Use
a clean applicator stick for each slide.
• Record the results

Blood group Anti –A Anti-B

A + -

B - +

AB + +

O - -

*From the above picture we conclude that :

(o) blood type is universal donor

(AB) blood type is universal acceptor (recipient)

Note that:

 Rh blood type is especially important for pregnant women. A

problem can occur when a woman who has Rh-negative blood
becomes pregnant with a baby (fetus) that has Rh-positive blood.
This is called Rh incompatibility.
 If the blood of an Rh-positive baby mixes with the blood of an Rh-
negative mother during pregnancy or delivery, the mother's
immune system makes antibodies. This antibody response is called
Rh sensitization and, depending on when it occurs, can destroy the
baby's red blood cells.
 Rh sensitization does not generally affect the health of the baby
during the pregnancy in which the sensitization occurs. But the
health of a baby with Rh-positive blood during a future pregnancy
is more likely to be affected. After sensitization has occurred, the
baby can develop mild to severe problems (called Rh disease or
erythroblastosis fetalis). In rare cases, if Rh disease is not treated,
the baby may die.
 An Rh test is done in early pregnancy to check a woman's blood
type. If she is Rh-negative, she can get a shot of Rh
immunoglobulin that almost always prevents sensitization from
occurring (injection with Rhogam that cause destruction of fetal
RhD+ red blood cells)
 Also Minor antigens (other than A, B, and Rh) on the red
blood cells are also checked for a match before a blood
transfusion may be found with an RBC antibody screen.
A blood type test is done:

•Before a person gets a blood transfusion.

•Before a person donates blood.

•Before a person donates an organ for transplantation.

•Before surgery.

•When a woman is planning to become pregnant or first becomes

pregnant.

•To show whether two people could be blood relatives.

•To check the identification of a person suspected of committing a crime.