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*- Immune respons : its characterized by the produuction of

proteins ( Igs) and specificially reactive
lymphocytes (T-cells ) when an animal
encounters a foreign macromolecules or cells.
antigens The inducing substances are called
i.e. antibody generators or immunogens.
*- Immunogenicity : it the inherent ability of the immunogen
(complete antigen ) to induce a specific immune
response and to react with the products of this
response (i.e. antibodies or the immune
reactive lymphocytes) .
*- Antigenicity : It is the ability of the foreign substance to react
with the products of that response .
Therefore, Antigens are the ligands that react with the products of an immune response
Also, the immunogenicity & antigenicity are two interchangeable terms which will be
used during discussion of the immune reponse during the period of this course.
In addition, HAPTEN HAS AN ANTIGENICITY but HAPTEN PLUS
PROTIEN CARRIER IS IMMUNOGEN
Overview of the Immune System
Immune System
Innate
(Nonspecific)
1
o
line of defense
Adaptive
(Specific)
2
o
line of defense
Protects/re-exposure
Cellular Components Humoral Components Cellular Components Humoral Components
Interactions between the two systems
Innate Immunity
Adaptive Immunity
Comparison of Innate and Adaptive
Immunity
*
No time lag
*
Not antigen specific
*
A lag period
*
Antigen specific
*
No memory developed
Memory developed
Functions of the Immune System
(Self/Non-self Discrimination)
• To protect from pathogens
• Intracellular (e.g. viruses and some bacteria
and parasites)
• Extracellular (e.g. most bacteria, fungi and
parasites)
• To eliminate modified or altered self
Infection and Immunity Balance
infection
immunity
Bolus of infection x virulence
immunity
Disease =
• Beneficial:
• Protection from Invaders
• Elimination of Altered Self
• Detrimental:
• Discomfort and collateral damage (inflammation)
• Damage to self (hypersensitivity or autoimmunity)
Effects of the Immune System
Innate (Nonspecific) Immunity
Innate Host Defenses Against Infection
• Anatomical barriers
– Mechanical factors
– Chemical factors
– Biological factors
• Humoral components
– Complement
– Coagulation system
– Cytokines
• Cellular components
– Neutrophils
– Monocytes and macrophages
– NK cells
– Eosinophils
Anatomical Barriers - Mechanical Factors
System or Organ Cell type Mechanism
Skin Squamous epithelium Physical barrier
Desquamation
Mucous Membranes Non-ciliated epithelium
(e.g. GI tract)
Peristalsis
Ciliated epithelium (e.g.
respiratory tract)
Mucociliary elevator
Epithelium (e.g.
nasopharynx)
Flushing action of
tears, saliva,
mucus, urine
Anatomical Barriers - Chemical Factors
System or Organ Component Mechanism
Skin Sweat Anti-microbial fatty
acids
Mucous Membranes HCl (parietal cells)
Tears and saliva
Low pH
Lysozymes and
Phospholipase A
Defensins (respiratory & GI
tract)
Antimicrobial
Sufactants (lung) Opsonin
Anatomical Barriers - Biological Factors
System or Organ Component Mechanism
Skin and mucous
membranes
Normal flora ☻Antimicrobial substances
☻Competition for nutrients
and colonization
Natural immune response (i-Humoral Components)
Component Mechanism
Complement
☻- Lysis of bacteria and some viruses
☻- Opsonin
☻- Increase in vascular permeability
☻- Recruitment and activation of phagocytic cells
☻- With the help of antibodies, they can destroy the
pathogens
Coagulation
system
☻- Increase vascular permeability
☻- Recruitment of phagocytic cells
☻- Β-lysine from platelets (a cationic detergent)
Lactoferrin and
transferrin
☻- Compete with bacteria for iron, therefore,
cause bacterial death.
Lysozymes
☻- Breaks down bacterial cell walls causing their lysis.
Cytokines (Interleukins
,
Interferon's
)
☻- They had various immunological effects
Interferons (IFNs )

They

are natural proteins produced by the cells of the immune system of most
vertebrates in response to challenges by foreign agents such as viruses, bacteria,
parasites and tumor cells. Interferons belong to the large
class of glycoproteins known as cytokines.
The discovery of interferon ;
, virologists , two Japanese smallpox for vaccine While aiming to develop an improved
, noticed that University of Tokyo working at the then Institute for Infection Disease at the
inactivated virus exhibited inhibited - with UV inoculated skin or testis previously - rabbit
- of the UV fractionation viral growth inhibitory factor, and began to characterise it by
ultracentrifuge using an homogenates irradiated viral .
Functions of inteferons :
Interferons in general have several effects in common .
► They are antiviral and possess antioncogenic properties


major lymphocyte activation, and enhancement of natural killer and Macrophage
classes I and II, and thus presentation of glycoprotein histocompatibility complex
T cells foreign (microbial) peptides to .


In a majority of cases, the production of interferons is induced in response to
microbes such as viruses and bacteria and their products (viral glycoproteins, viral
RNA, bacterial endotoxin, bacterial flagella, CpG DNA), as well as mitogens and
interleukin other cytokines, for example 1 , interleukin 2 , - interleukin 12 tumor ,
, that are synthesised in the response stimulating factor - colony and necrosis factor
to the appearance of various antigens in the body .
Their metabolism and excretion take place mainly in the liver and kidneys. They rarely
brain barrier - blood and the placenta pass the
Type I interferons
- B , NK cells ) lymphocytes β are secreted by many cell types including - α and IFN - IFN
), macrophages, fibroblasts, endothelial cells, osteoblasts and others. cells - T and cells
viral response, and - and NK cells to elicit and anti macrophages They stimulate both
at the site of viral leukocytes ω is released by - . IFN tumors are also active against
infection or tumors .

Type II interferons
is involved in the regulation of the immune and inflammatory responses; in γ - IFN
humans, there is only one type of interferon-gamma. It is produced in activated T-cells
tumor effects, but these are - viral and anti - γ has some anti - . IFN natural killer cells and
generally weak .
Th γ released by - potentiates the effects of the type I IFNs. IFN cytokine However, this 1
to a site of infection, resulting in increased inflammation. It leukocytes recruits cells
γ released - to kill bacteria that have been engulfed. IFN macrophages also stimulates
by Th 1 cells is also important in regulating the
Th 2 response. As IFN-γ is vitally implicated in the regulation of immune response, its
autoimmune disorders can lead to

production
Autoimmune diseases
i-What are autoimmune diseases?
Our bodies have an immune system that protects us from disease and infection. But if
you have an autoimmune disease, your immune system attacks itself by mistake, and
you can get sick. Autoimmune diseases can affect connective tissue in your body (the
tissue which binds together body tissues and organs). Autoimmune disease can affect
many parts of your body, like your nerves, muscles, endocrine system (system that
directs your body’s hormones and other chemicals), and digestive system.
Autoimmunity is the failure of an organism to recognize its own constituent
parts (down to the sub-molecular levels) as "self", which results in an immune
response against its own cells and tissues. Any disease that results from such an
aberrant immune response is termed an autoimmune disease. Autoimmune diseases,
therefore are a large group of diseases characterized by abnormal functioning of the
immune system that causes your immune system to produce antibodies against your
, 1 Diabetes Type , Crohn's disease the prominent examples being - own tissues
and Sjögren's syndrome (SLE), Systemic Lupus Erythematosus , Coeliac disease
(RA). Rheumatoid arthritis
Prognosis of Autoimmune diseases
Although autoimmune diseases are chronic, the course they take is unpredictable.
A doctor cannot foresee what will happen to the patient based on how the disease
starts. Patients should be monitored closely by their doctors so environmental factors
or triggers that may worsen the disease can be discussed and avoided and new medical
therapy can be started as soon as possible. Frequent visits to a doctor are important in
order for the physician to manage complex treatment regimens and watch for medication
side effects.
Who is at risk for getting autoimmune diseases?
Most autoimmune diseases occur in women, and most often during their childbearing years.
Some of these diseases also affect African American, American Indian, and Latina women more
than white women. These diseases tend to run in families, so your genes, along with the way
your immune system responds to certain triggers or things in the environment, affect your
chances of getting one of these diseases. If you think you may have an autoimmune disease,
ask your family members if they have had symptoms like yours. The good news is that if you
have an autoimmune disease, there ARE things you can do to feel better!
What are the most common symptoms of autoimmune diseases?
There are more than 80 types of autoimmune diseases. Learning the symptoms of some of the
more common autoimmune diseases can help you recognize the signs if you get one. But some
autoimmune diseases share similar symptoms. This makes it hard for doctors to find out if you
really have one of these diseases, and which one it might be. This can make your trip to doctors
long and stressful. The most common symptoms of the autoimmune diseases include
tiredness, depression , sensitivity to cold, weight gain, muscle weakness and cramps, dry hair
tough skin, constipation and sometimes there are no symptoms
Natural immune response
ii-Cellular Components
Cell Functions
Neutrophils ☻-Phagocytosis and intracellular killing
☻- Inflammation and tissue damage
Macrophages ☻- Phagocytosis and intracellular killing
☻- Extracellular killing of infected or altered self
targets
☻- Tissue repair
☻- Antigen presentation for specific immune
response
NK and LAK cells ☻- Killing of virus-infected cells and altered self
targets
Eosinophils ☻- Killing of certain parasites
Phagocytosis
and
Intracellular Killing
Phagocyte Response to Infection
• The Signals
–N-formyl methionine-containing
peptides
–Clotting system peptides
–Complement products
–Cytokines released by tissue
macrophages
• Phagocyte response
–Vascular adherence
–Diapedesis
–Chemotaxis
–Activation
–Phagocytosis and killing
Phagocytosis
Steps of Phagocytosis
•Attachment
•Pseudopod extension
•Phagosome formation
•Granule fusion
•Phagolysosome formation
Attachment via Receptors:
IgG FcR
ScavengerR
Complement R
Toll-like R
Initiation of Phagocytosis
☻-
Characteristic nucleus and cytoplasm

-
specific granules
☻-
CD 66 membrane marker
Phagocytes - Neutrophils (PNMs)
• Characteristic nucleus
• Lysosomes
• CD14 membrane marker
Phagocytes - Macrophages
Natural Killer (NK) cells
Also known as large
granular lymphocytes (LGL)
Kill virus-infected or
malignant cells
Identified by the presence of
CD56 & CD16 and absence
of CD3
Activated by IL2 and IFN-γ
to become LAK cells
What are Natural Killer Cells?
Natural killer (NK) cells are an important first line of defense against newly arising malignant cells and
cells infected with viruses, bacteria, and protozoa. They form a distinct group of lymphocytes with no
immunological memory and are independent of the adaptive immune system. Natural killer cells
constitute 5 to 16 percent of the total lymphocyte population. Their specific function is to kill infected and
cancerous cells (AAA Reference Laboratories, Inc .). Most of us have enough natural killer cells (cell
counts) in our body, however many of us don't have enough natural killer cells that are active. These
inactive natural killer cells are present in great numbers in our blood, lymph nodes, organs, and tissue,
but they are not killing foreign invaders such as infectious organisms and malignant cells that constantly
affect all of us
.
What You Should Know about Natural Killer Cells Activity?
It is known that:
Almost all cancer patients have very low levels of natural killer cell activity: usually 0 to 20
have low levels in Chronic Fatigue Syndrome Many patients with chronic diseases including Fibromyalgia and
the range of 10 to 30.
A wide variety of Auto Immune Disorders including Rheumatoid Arthritis, Lupus, Multiple Sclerosis and
others have low levels in the 10 to 30 range
Most patients with chronic and/or recurrent infections (such as Staph, Sinusitis, Bronchitis, Tonsillitis,
Pneumonia, and ear infections, etc.) have low levels in the 10 to 50 range
Many patients with symptomatic EBV, CMV, HPV and other chronic viral infections are in the 0-20 range
It is also known that, there is a direct age related decrease in natural killer cell activity from 20 to 80
years
of age which may partially explain why the risk of cancer increases with each decade of life
Low natural killer cell activity is a significant independent risk factor for the future development of cancer
,as well as other chronic diseases and illnesses.
Also, low natural killer cell activity is a strong predictor of poor prognosis of survival for cancer patients.
Therefore, the higher the natural killer cell activity in patients with cancer the better their prognosis is for
survival
Non-specific Killer Cells
NK and LAK cells
ADCC (K) cell
Activated macrophages
Eosinophils
They all kill foreign
and altered self
targets
Toxic compounds are : Superoxide anion (O
2
-
), Hydrogen peroxide
(H
2
O
2
), Singlet oxygen (
1
O
2
) and Hydroxyl
radical (OH*)
Intracellular Killing Pathways
a-Respiratory Burst
a-1- Oxygen-Dependent Myeloperoxidase-Independent Reactions
Pentose-P + NADPH
G-6-P-dehydrogenase
Glucose +NADP
+
NADPH oxidase
Cytochrome
b558
NADP
+
+ O
2
-
NADPH + O
2
Superoxide dismutase
H
2
O
2
+
1
O
2
2O
2
-
+ 2H
+
2O
2
-
+ H
2
O
2
OH* + OH
-
+
1
O
2
Respiratory Burst (continued)
a-2-
Oxygen-Dependent Myeloperoxidase-Dependent Reactions
myeloperoxidase
OCl
-
+ H
2
O H
2
O
2
+ Cl
-
2OCl
-
+ H
2
O
1
O
2
+ Cl
-
+ H
2
O
Toxic compounds: Hypochlorous acid (OCl
-
), and Singlet oxygen (
1
O
2
)
Respiratory Burst (continued)
Detoxification Reactions
H
2
O
2
+ O
2
Superoxide dismutase
H
2
O + O
2
Catalase
2O
2
-
+ 2H
+
2 H
2
O
2
Effector Molecule Function
b-Oxygen-Independent Killing in
the Phagolysosome
Cationic proteins (cathepsin)
Damage to microbial
membranes
Lysozyme
Hydrolyses mucopeptides
in the cell wall
Lactoferrin
Deprives pathogens of iron
Hydrolytic enzymes (proteases) Digests killed organisms
Summary of Intracellular Killing
Pathways
Intracellular Killing
Oxygen
Dependent
Oxygen
Independent
Myleoperoxidase
Dependent
Myleoperoxidase
Independent
Nitric Oxide Dependent Killing
TNF
TNF
Nitric Oxide
Nitric Oxide
Lymphokine Activated Killer (LAK)
cell
kills
malignant
cells
kills
transformed
and malignant
cells
Regulation of NK Cell Function
•MHC I •KIR •KAR •KAL
•No Killing •Killing
Their metabolism and excretion take place mainly in the liver and kidneys. They rarely
brain barrier - blood and the placenta pass the
Type I interferons
- B , NK cells ) lymphocytes β are secreted by many cell types including - α and IFN - IFN
), macrophages, fibroblasts, endothelial cells, osteoblasts and others. cells - T and cells
viral response, and - and NK cells to elicit and anti macrophages They stimulate both
at the site of viral leukocytes ω is released by - . IFN tumors are also active against
infection or tumors .

Type II interferons
is involved in the regulation of the immune and inflammatory responses; in γ - IFN
humans, there is only one type of interferon-gamma. It is produced in activated T-cells
tumor effects, but these are - viral and anti - γ has some anti - . IFN natural killer cells and
generally weak .
Th γ released by - potentiates the effects of the type I IFNs. IFN cytokine However, this 1
to a site of infection, resulting in increased inflammation. It leukocytes recruits cells
γ released - to kill bacteria that have been engulfed. IFN macrophages also stimulates
by Th 1 cells is also important in regulating the
Th 2 response. As IFN-γ is vitally implicated in the regulation of immune response, its
autoimmune disorders can lead to

production
Autoimmune diseases
Autoimmunity is the failure of an organism to recognize its own constituent parts
(down to the sub-molecular levels) as "self", which results in an immune response
against its own cells and tissues. Any disease that results from such an aberrant
immune response is termed an autoimmune disease. Autoimmune diseases, therefore
are a large group of diseases characterized by abnormal functioning of the immune
system that causes your immune system to produce antibodies against your own
Coeliac , 1 Diabetes Type , Crohn's disease the prominent examples being - tissues
Rheumatoid and Sjögren's syndrome (SLE), Systemic Lupus Erythematosus , disease
(RA). arthritis
Prognosis of Autoimmune diseases
Although autoimmune diseases are chronic, the course they take is unpredictable. A
doctor cannot foresee what will happen to the patient based on how the disease
starts. Patients should be monitored closely by their doctors so environmental factors
or triggers that may worsen the disease can be discussed and avoided and new
medical therapy can be started as soon as possible. Frequent visits to a doctor are
important in order for the physician to manage complex treatment regimens and
watch for medication side effects
.
What are autoimmune diseases?
Our bodies have an immune system that protects us from disease and infection. But if
you have an autoimmune disease, your immune system attacks itself by mistake, and you
can get sick. Autoimmune diseases can affect connective tissue in your body (the tissue
which binds together body tissues and organs). Autoimmune disease can affect many
parts of your body, like your nerves, muscles, endocrine system (system that directs your
body’s hormones and other chemicals), and digestive system.
Who is at risk for getting autoimmune diseases?
Most autoimmune diseases occur in women, and most often during their childbearing
years. Some of these diseases also affect African American, American Indian, and Latina
women more than white women. These diseases tend to run in families, so your genes,
along with the way your immune system responds to certain triggers or things in the
environment, affect your chances of getting one of these diseases. If you think you may
have an autoimmune disease, ask your family members if they have had symptoms like
yours. The good news is that if you have an autoimmune disease, there ARE things you
can do to feel better!
What are the most common symptoms of autoimmune diseases?
There are more than 80 types of autoimmune diseases. Learning the symptoms of some
of the more common autoimmune diseases can help you recognize the signs if you get
one. But some autoimmune diseases share similar symptoms. This makes it hard for
doctors to find out if you really have one of these diseases, and which one it might be.
This can make your trip to doctors long and stressful. The most common common
symptoms of the autoimmune diseases include tiredness depression
sensitivity to cold weight gain muscle weakness and cramps dry hair tough skin
constipation sometimes there are no symptoms
What are Natural Killer Cells?
Natural killer (NK) cells are an important first line of defense against newly arising malignant cells and
cells infected with viruses, bacteria, and protozoa. They form a distinct group of lymphocytes with no
immunological memory and are independent of the adaptive immune system. Natural killer cells
constitute 5 to 16 percent of the total lymphocyte population. Their specific function is to kill infected and
cancerous cells (AAA Reference Laboratories, Inc .). Most of us have enough natural killer cells (cell
counts) in our body, however many of us don't have enough natural killer cells that are active. These
inactive natural killer cells are present in great numbers in our blood, lymph nodes, organs, and tissue,
but they are not killing foreign invaders such as infectious organisms and malignant cells that constantly
affect all of us
.
What You Should Know about Natural Killer Cells Activity?
It is known that:
Almost all cancer patients have very low levels of natural killer cell activity: usually 0 to 20
have low levels in Chronic Fatigue Syndrome Many patients with chronic diseases including Fibromyalgia and
the range of 10 to 30.
A wide variety of Auto Immune Disorders including Rheumatoid Arthritis, Lupus, Multiple Sclerosis and
others have low levels in the 10 to 30 range
Most patients with chronic and/or recurrent infections (such as Staph, Sinusitis, Bronchitis, Tonsillitis,
Pneumonia, and ear infections, etc.) have low levels in the 10 to 50 range
Many patients with symptomatic EBV, CMV, HPV and other chronic viral infections are in the 0-20 range
It is also known that, there is a direct age related decrease in natural killer cell activity from 20 to 80
years
of age which may partially explain why the risk of cancer increases with each decade of life
Low natural killer cell activity is a significant independent risk factor for the future development of cancer
,as well as other chronic diseases and illnesses.
Also, low natural killer cell activity is a strong predictor of poor prognosis of survival for cancer patients.
Therefore, the higher the natural killer cell activity in patients with cancer the better their prognosis is for
survival
Complement: History
Discovered in 1894 by
Bordet
It represents lytic activity
of fresh serum
Its lytic activity destroyed
when heated at 56C
for 30 min
Complement functions
• Host benefits:
– Opsonization to enhance phagocytosis
– Phagocyte attraction and activation
– Lysis of bacteria and infected cells
– Regulation of antibody responses
– Clearance of immune complexes
– Clearance of apoptotic cells
• Host detriments:
– Inflammation, anaphylaxis
Proteins of the complement
system (nomenclature)
• C1(qrs), C2, C3, C4, C5, C6, C7, C8, C9
• factors B, D, H and I, properdin (P)
• mannose binding lectin (MBL), MBL associated
serine proteases (MASP-1 MASP-2)
• C1 inhibitor (C1-INH, serpin), C4-binding
protein (C4-BP), decay accelerating factor
(DAF), Complement receptor 1 (CR1), protein-
S (vitronectin)
• C-activation: alteration of C proteins such that they
interact with the next component
• C-fixation: utilization of C by Ag-Ab complexes
• Hemolytic units (CH50): dilution of serum which
lyses 50% of a standardized suspension of Ab-coated
r.b.c
• C-inactivation: denaturation (usually by heat) of an
early C-component resulting in loss of hemolytic activity
• Convertase/esterase: altered C-protein which acts
as a proteolytic enzyme for another C-component
Definitions
Activation product of complement
proteins (nomenclature)
When enzymatically cleaved, the larger moiety,
binds to the activation complex or membrane
and the smaller peptide is released in the
microenvironment
Letter “b” is usually added to the larger,
membrane-binding, peptide and “a” to the
smaller peptide (e.g., C3b/C3a, C4b/C4a,
C5b/C5a), EXCEPT C2 (the larger, membrane-
binding moiety is C2a; the smaller one is C2b)
Activated component are usually over-lined: e.g.
C1qrs
Pathways of complement
activation
CLASSICAL
PATHWAY
ALTERNATIVE
PATHWAY
activation
of C5
LYTIC ATTACK
PATHWAY
antibody
dependent
LECTIN
PATHWAY
antibody
independent
Activation of C3 and
generation of C5 convertase
Components of the Classical
Pathway
C4
C3
C1 complex
Classical Pathway
Generation of C3-convertase
Classical Pathway
Generation of C3-convertase
C4b
_____
C4b2a is C3 convertase
Classical Pathway
Generation of C5-convertase
C4b
C3b
________
C4b2a3b is C5 convertase;
it leads into the Membrane
Attack Pathway
54
Biological Activities of Classical
Pathway Components
Component Biological Activity
C2b Prokinin; cleaved by plasmin to yield kinin, which
results in edema
C3a Anaphylotoxin; can activate basophils and mast
cells to degranulate resulting in increased vascular
permeability and contraction of smooth muscle cells,
which may lead to anaphylaxis
C3b Opsonin
Activation of phagocytic cells
C4a Anaphylaotoxin
C4b Opsonin
C1-inhibitor deficiency:
hereditary angioedema
Components of mannose-binding
lectin pathway
MBL MASP1
Mannose-binding lectin pathway
MBL
_____
C4b2a is C3 convertase; it
will lead to the generation of
C5 convertase
MASP1
Components of the
alternative pathway
C3
Spontaneous C3 activation
C3
i
Generation of C3 convertase
C3iBb complex has a very short half life
b
C3b
b C3b
If spontaneously-generated
C3b is not degraded
C3-activation
the amplification loop
C3 b
C3b
C3
b
C3-activation
the amplification loop
C3b
b
General Introduction
◙ The immune system is a set of mechanisms that protect an organism from
pathogens by identifying and killing infection .
◙ parasitic worms to viruses task is extremely difficult, since pathogens range from his T
tissues and cells and these diverse threats must be detected with absolute specificity amongst normal .
◙ new ways to avoid detection by the immune evolving Pathogens are also constantly
hosts system and successfully infect their .
◙ To meet this challenge, multiple mechanisms have evolved to recognize and neutralize pathogens .
◙ pattern , defensins called antimicrobial peptides These mechanisms include
complement system , and the recognition receptors .
◙ More sophisticated mechanisms, however, developed relatively recently, with the
humans . The immune systems of vertebrates such as vertebrates evolution of
, and tissues, which interact in an organs , cells, proteins consist of many types of
elaborate and dynamic network .
◙ As part of this more complex immune response, the vertebrate system adapts over
time to recognize particular pathogens more efficiently .
◙ and allows even immunological memories The adaptation process creates
more effective protection during future encounters with these pathogens.This
vaccination is the basis of acquired immunity process of .

General Introduction (continued)
◙Disorders in the immune system can cause diseases.
◙Immunodeficiency diseases occur when the immune system is less active
than normal, resulting in recurring and life-threatening infections.

◙Immunodeficiency can either be the result of a genetic disease, such as severe
combined immunodeficiency, or be produced by pharmaceuticals or an infection,
such as the acquired immune deficiency syndrome (AIDS) that is caused by the
retrovirus HIV .
◙In contrast, autoimmune diseases result from a hyperactive immune system
attacking normal tissues as if they were foreign organisms. Common
autoimmune diseases include rheumatoid arthritis, diabetes mellitus type 1 and lupus
erythematosus.
◙ Therefore, immunity or the resistance is the sum of all naturally occurring and
acquired defense mechanisms that protect the organism from infectious
diseases.and the study of this mechanisms that a host has evolved to get rid itself
of pathogens and other foreign substances.
◙The immune system so, has at least three major functional properties that
distinguish it
from all the body's other defenses:
.
Immunity (resistance):
It the sum of all naturally occurring defense mechanisms
that protect the organism (or host) from infectious
diseases. In addition, it include the study of the
mechanisms that a host has evolved to get rid itself from
the invading pathogens and other foreign substances.
The immune system so, has at least three major
functional properties that distinguish it from all the body's
other defenses:
The first: Is its extreme specificity, the ability to
recognize and distinguish among a large or
vast number of different target molecules, and
to respond (or not respond) to each of these
individually
Second: The immune system discriminates between self
(body ingredients ) and non self ( foreign bodies),
so that it normally coexists peacefully with all
of the immunerable proteins and other organic
materials that make up the host but responds
vigorously against foreign substances,
including cells or tissues from other people .
Third: The immune system has memory, that is, the ability
to be molded by its experiences so that subsequent
encounters with a particular foreign pathogen
provoke more rapid and more vigorous responses
than occurred at the initial encounter.
A- Non a specific or innate immune response:
This consists of the pre-existing defenses of an
animal, such as barrier layers and secretions.
It has the following properties:
i- It does not exhibit high specificity.
ii- It does not depend on a complete (specific)
recognition of the antigen.
iii- A single mechanism protect against many
pathogen.
B- Specific or adaptive immune response:
This response involves the cells of the
immune system and frequently leads to a state
of immune memory, and finally destroying the
invading organisms.
Comparison between the non-specific and the specific
immunity
Specific Immunity Non-specific Immunity
Response is antigen-
dependent (antigen-specific)
Response is antigen-
independent (Not antigen-
specific)
There is a lag time between
exposure and maximal
response
There is immediate maximal
response
Exposure to the Pathogen
produce immunological
memory
Exposure to the Pathogen did
not produce immunological
memory
2- The Non-Specific (Innate Immune)
Response
1- First defense line:
a-Anatomical barriers:
- Skin which physically preventing the interaction
between the host and the pathogen.
- Intestinal movement and mucus coating their walls.
- Oscillation of broncho-pulmonary cilia.
b-secretory molecules:
-These secretions include organic acids in skin secretions,
thiocyanate in saliva, low molecular weight fatty acids, bile acids
in lower gastric intestinal tract, transferring, lactoferrin,
lyzozyme, interferons, fibronectin, complement, etc. in serum,
interferons and tumor necrosis factor at the site of inflammation.
2. Second defense line:
They represent the Cellular components, and they include
phagocytic cells either polynuclear phagocytes or mononuclear
phagocytes and NK cells.
Polynuclear phagocytes:
Neutrophils (Polymorph nuclear cells PMNs) are the most important
cellular components in bacterial destruction. They are relatively large
and most abundant white blood cells (65% of leucocytes) with lobed
nucleus and cytoplasmic granules (lysosomes
All phagocytic cells have receptors for a variety of molecules. Most
pertinent to non-specific immune response are receptors for IgG-Fc,
complement, interferon, TNF and certain bacterial components.
Receptor interactions with these ligands promote phagocytosis and
activation for efficient killing of pathogens
The figure shows a Neutrophil in a blood film
Example of Phagocytosis: A macrophage
attacking E.coli
Chemotactic response to inflammatory stimulus
And the steps of this type of response
1- Adherence 2- Diapedesis 3- going to the inflammatory site 4- Re-activation of
adherence via histamine and thrombin secretions.
Histopathology of bladder shows eggs of
Schistosoma haematobium surrounded by intense
infiltrates of eosinophils
NK cells and their activation
3-Front defense line
:
The major physiologic roles of natural killer cell (NK cells) appear to be in
the early host defense against microbial agents. Nk cells, therefore, help to
protect against a range of infections before the T-cell and B-cell response
have developed. NK cells may thus function as a bridge between the innate
and the acquired immune systems, acting as a front line of defense , while
producing cytokines to promote the development of a specific immune
response.
1- Derived from bone marrow.
2- Lack most markers for T and B cells
(no TCR or CD3).
3- Don’t undergo thymic maturation.
4- Express CD56, a specific NKs marker
5- Express a low affinity receptor for Fc
portion of IgG called FcR (CD16),
also expressed on granulocytes and
macrophages.
6-Cytokines especially IL -2 promotes further
differentiation in to lymphokine – activated
killer cells (LAK).
Acute-phase Response
Most soluble mediators of innate immune response are
found in relatively small amounts, with the exception of C3,
in the serum under normal conditions.
The concentrations of several of these proteins, however,
can increase as much as 1000-fold during serious
infections, as part of accordinated protective reaction called
the hepatic acute-phase-response. In this response, the
liver temporarily increases its synthesis of more than
adozen different serum proteins that participate in anti-
microbial defense, including complement factors C3 and B,
the mannose binding proteins, C-reactive protein, serum
amyloid protein P, and others.
The response occurs when hepatocytes are exposed to
certain cytokines
3.1 Cells of the immune system
Immunity (resistance): It the sum of all naturally occurring
defense mechanisms that protect human from infectious disease
Non – specific
( Innate )
- Mucous membranes
- Phagocytic cells
- Enzymes in secretion
- -Interferons ( α,β,γ)
- - NKCs
- -Skin
- . Macrophages
Specific
( Acquired )
Naturally acquired
- Placental transfer of antibodies( Passive )
- Recovery from disease ( Active )
- Administration of antitoxin ( Passive )
- Vaccinations ( Active )
Artificially acquired
Natural ( Innate )
Specific ( Adaptive )
or
(Acquired)
Less specific . -
- Skin & mucous membrane .
-NK cells .
- Complement cascade .
- Phagocytosis .
- C- reactive protein .
Active Passive
- -Induced by contact with foreign antigens .
- - Consist of clinical infection , immunization with live or
- killed infectious agents or their toxins .
- - Long term.
- - Induced by antibody performed in
- another host
- - Ab injected in the incubation period
- Short term .
- with the innate or inherent is which mechanism physiologic This is a
following properties
It does not exhibit specificity
Do not depends on specific
recognition of a foreign
material
A single
mechanisms
Protect
Against
many
paths
Artificially acquired
Naturally acquired
active
passive
passive active
First:
Non – specific Immunity ( Innate)
:-
OWN IMMUNITY Definition :- the body forms his when
stimulated (sensitized ) by introduction of
immunogenic agent.
Natural Infection
Types
*living attenuated vaccine * killed vaccine .
Artificial bacterial products
*Endotoxins.
* Exotoxins.
Others .
Characters :- * slowly developed .
*longer duration
(and leave a potential immunity , so there is A rapid response
in the future to the Same antigen ) leads to ??
*-Homogenous antibodies
*- Cellular defense mechanism play a role
Mechanism of Acquired immunity :-
Humeral
A b
T_Cells
Cellular
classification of acquired Immunity:-
- passive Acquired Immunity :-
Definition: acquired Immunity by given already form antibodies or antitoxic serum or gamma
globulins from normal or convalescent individuals or Trans placental or lactation .
Trans placental .
Natural
Types Lactation (Colostrum).
Antitoxin serum tetanus. (Anti_ cobra venom)
Artificial
Gamma globulins.
Rapidly developed . - * - : characters -
* -Short duration .
[ Rapidly eliminated in 2-4 WKS due to the formation of anti – antibodies (a disadvantage )].
*-Heterogeneous antibodies .
* -Cellular mechanism not stimulated .
(No memory ).
- : Side effects - *
*- Hyper sensitivity reactions against the
foreign serum
*-Neurological affection in some cases
( Encephalitis ).
*-Superadded in infections
e.g. (AIDS & HEPAT) .
- Cellular immunity .
- Cell mediated immunity .
- (T- lymphocytes-Mediated)
- Humoral immunity
Antibody mediated immunity.
( B- lymphocyte)
B- lymphocyte
(Protection is mediated by
the produced antibodies)
Helper T- LYMPH .
Cytotoxic T-lymphocyte
TH2
TH1
help
CD4
CD8
help
Haematopoietic stem cell
Myeloerythroid progenitor
Lymphoid stem cell
B-lymphocyte
NK cell
T-lymphocyte
monocyte neutrophil eosinophil macrophage basophile RBC platelets
Antibody
L-CHAIN
H-CHAIN
Immunoglobulins or Humoral antibodies
* B-
Lymphocytes
to proliferate , stimulated Lymphocytes are - * Upon exposure to antigen , B
differentiate and mature into LARGE PLASMA CELLS (large lymphocytes)
*
The large mature B-Lymphocytes have
short life span (
days to weeks
) .
This cell type consists ( 20 – 25%) of the total peripheral lymphocytes
n mammals , they mature in bone marrow , then, migrate to secondary
lymphoid organs ( e.g. spleen & Lymph nodes ) .
Secreted
This type of cells is
involved in the
* Some large mature B-Lymphocytes (B- cells ) can be converted
long life span cells which have - into small B
And serve as Memory cells
Secondary
Immune
Response
Activation & differentiation of B-Lymphocytes , in certain instances ,
needs a Helper T- Lymphocytes activity to enhance the above to
processes in that B-Lymphocytes.
*- THEY CONSTITUTE 65 -80 10 of total peripheral lymphocytes .
*- They have long life span ( months to years ).
*- They mature in thymus gland before migrating to lymphoid organs
*- Upon exposure to antigen , T -cell proliferate .
How ever , their specific effectors molecules are not secreted
and remains firmly Attached to their cellular membranes
Giving what is called
cell-mediated immune response
*- They are involved in a variety of cell-mediated immunological responses
T-Lymphocytes :-
defense
against
malignant
cells
graft rejection
bacteria & protozoa
Fungi
hyper sensitivity
reactions
viruses
T-CELLS
T-HELPER (TH) T-SUPRESSOR(TS) T-CYTOTOXIC (TCs) T-DELAYED
T- Helper :Their Surface Antigen : is T4 (CD4) .
*They Promote Maturation Of Antigen .
*Stimulated B and T cells.
And
Enhance their response
T – suppressor cells: * Their Surface antigen is T8(CD8).
* they suppress the effect of T – helper cells .
i.e.
*Suppress T &B – response .
T –cytotoxrc: * their Surface antigen T8(CD).
* they specifically destroy target cells.
virus infected cells unacceptable grafted cells
cells tumor
T – delayed hypersensitivity & T cell mediated immunity.CD4 (T4)
*they are responsible for delayed hypersensitivity reactions to different
antigens , particularly those of intra cellular parasites & contact allergen .
In general : * some of the stimulated T-cells release soluble substances
lymphokines that modulate the behavior of other cells.
Helper-
Sensitivity
Cells ( TdH)
and
T-CELL MEDIATED
IMMUNITY
(Tcmi )
*- most antigens which have a small number of epitopes and require carrier need
T – cell cooperation with B- cells
for antibodies production .
* Deficiency of B – cells (and\or) T-helper cells
leads to defective synthesis of antibodies.
* its over activity lead to
the majority of B-lymphocytes express both surface IgM & IgD, very few express
surface IgG & IgA or IgE in the circulation.
*the majority of B-cells also carry class 2 major histocompatibility complex )class П
MHC) products which are functionally important in
Regulation of immune response
Autoimmune disorders
2- T cell Activation
When a T cell encounters an antigen presenting cell (APC), the specificity of
its TCR determines the outcome.
Only if the TCR recognizes its particular antigen MHC combination does
activation occur.
The recognition of appropriately presented antigen activates T cells to
proliferate, differentiate and perform their effector functions.
Activation of helper T-cells leads to the production of lymphokines that
promote cellular and humoral immune responses, whereas activation of
cytotoxic T cells results in killing of the antigen bearing cells.
Co -operation of innate & specific Immunity
in
Host defense against infection
*Antibodies promote Phagocytosis or activate complement to kill microbes
*T-lymphocytes enhance phogocytic and microbial functions of macrophages
+
INNATE IMMUNITY
BACTERIA PHOGOCYTE PHOGOCYTE
INEFFECTIVE
BACTERIA
+
SERM
COMPLEMENT
BACTERIA
+
complement
SPECIFIC IMMUNITY
Bacterial lyses
PHOGOCYTE
B-Lymph
Opsonization
And Phagocytosis
T-Lymph
+
B-Lymph
Cell
Mediated
response
a
b
s
LYSIS
A b BACTERIA
+
bacteria
Direct lyses
BACTERIA
In direct lyses by C.
Embryo Liver stem cell In Bone marrow
+
A 9
+
A 9
memory cells
HUMORAL ANTIBODIES
PLASMA
CELLS
central or
primary
lymphoid
organs
(tissues)
B_Cells T_Cells
Secondary
Lymphoid
Organs
Spleen or
Bone marrow
T B
Effector
Killer
cells
Specific memory and self-limitation of Immune response
Primary Anti A
RESPONSE
Ag A
infection
Secondary anti A
response
weeks
12 weeks
*- Antigen enhance THE production of specific Antibody A.
*- the secondary response to Ag A is more rapid and larger
then the primary response ( memory cells ) .
*- Antibodies Titer decline ( with time ) after each immunization .
Serum
AB
Specific immune response :
agents capable of It is developed as a result of exposure to a variety of
inducing an immune response
( i.e. immunogens )
Macro molecules microbes that colonize vaccines
in the body
Hapten is a micomolecule may be conjugate with a carrier protein in the
blood to be immunogen (antigen)
Specific immune response
Humoral cellular
B. Cells T-CELLS
*- They are two interrelated & interdependent mechanisms .
in the diet
in the form of hapten A special case Antigen
Specific immune response can be further
Classified according to its components into
primary secondary
Initial exposure to a particular on farther or
Infectious agent or immunogen repeated exposure
Induction phase of lymphocytes to antigen ( same )
proliferation T-CELLS
PLASMA CELLS increased B-CELLS
resistance
develops
through
Antibodies
humoral
Sensitized T-CELLS
Cellular Immune response
Humoral
response
Cellular
response
Acquired immune response
Has both good ( desirable ) and
Bad ( undesirable ) consequence
undesirable
Desirable
Immune
response
Protection
From infections
agents
Control of
Pre-cancerous
growths
Allergies (hypersensitivity)
Autoimmune diseases
Graft rejection
Interactions & functions of the major
components of the immune system
ANTIBODY – MEDIATED
IMMUNE RESPONSE
Main defense against
* exteracellular, encapsulated
pathogenic bacteria
e.g. streptococci & staphylococci
• *Neutralizations of toxins e.g.(
tetanus)
• * viruses ) Hepatitis C,A,B…….(
CELL MEDIATED REPONSE
Two major components
Cytotoxic
T-CELLS
Viruses
Acts by
Destroying
Virus- infected
cells
T-HLPER & MACROPHAGES
Intracellular bacteria
* ( mycobacterium &tuberculosis)
* Fungi
B-CELLS
T-CELLS
HELPER
CD4
LYMPHOKINES
CYTOTOXIC
CD8
PLASMA CELL
ANTIBODIES
+
COMPLEMENT
+
NEUTROPHILS
KILLING OF
BACTERIA
IL-2,IL-4.IL-5
IL_2
ACTIVATED HELPER
AND MACRO PHAGES
INHIBIT
INTRACELLULAR
Bacteria
&
fungi
Activated
Cytotoxic cells
Kill
Virus – infected
cells
IL_2
Neutralize
Toxins
Defense mechanism against viral infection
VIRUS ANTIGEN
VIRUS
VIRUS infection
cell
MHC Class I
T-Cell
receptor
virus
IgM
CD8
CYTOTOXIC
B-CELLS
INTERLEUKIN-4
INTERLEUKIN-5
MHC
Class II
T-Cell
RECEPTOR
CD4
( T_HELPER )
Killing
*- Recognition of phases :- antigen recognition ( binding of Ag to specific
receptor on mature lymphocyte ( exist prior to ag exposure )
*- activation proliferation & differentiation of lymphocytes is the sequence
of events induced in lymphocytes as result of Ag recognition .
*- Effectors phases elimination of antigen [ is the stage of the response
At which the sensitized cells perform the function that (eliminate of Ag)
Some antigen – stimulated lymphocytes die by process called programmed cell
death ( apaptosis ).
T
B
OR + Ag
NATIVE
LYMPHOCYTES
Recognition
phase
ACTIVATION
PHASE
programmed cell
Death
Effector
phase
Elimination
OF Ag
Phagocytosis
complement
Immunogenicety ability to induce immune response
Antigenicity ability of the substance to react specifically with
immune system
Happen is incomplete antigen ( di nitro phenol or penicillin)
It cannot stimulate humoral or cellular reactions but can react with these
products specifically so it is Antigenic not immunogenic
If they reacted with larger carrier protein (e.g., albumin , globulin or
synthetic poly peptide ) . It will be Immunogenic
Animals injected with this hapten – protein
Complex will make antibodies to this hapten ,
Only if it is ( hapten ) covalently linked to
the carrier (chemically bonded)
must be
Antigenic
Immunogenic
are not necessary to be
ANTIBODY
HAPTEN CARRIER
PROTEIN
PRTOCAL
NO
Anti carrier only
Anti carrier only
Anti carrier
&
Anti hapten
YES
NO
YES
YES
NO
YES
YES
( not chemically linked)
YES
(CHEMICALLY LINKED )
i
II
III
IV
Immune response :- its characterized by the production of proteins
called immunoglobulins( Igs) and specificially reactive lymphocytes (T-
cells ), which carry their own effector molecules on their surfaces, when
an animal encounters a foreign macromolecules or cells .
erators ) gen body anti i.e ( antigens The inducing substances are called
or immunogens
*- Immunogenicity & antigenicity : Interchangeable terms used during
discussion of the immune reponse.
*- Immunogenicity : it the inherent ability of asubstance ( Immunogen (
complete antigen ) to induce a specific immune response and to react with
the product of this response .
*- Antigenicity : the ability of the inducing substance (Antigen) to react
with the products of the immune response (i.e. the antibodies and|or the
effector molecules of the T-lymphocytes). .
HAPTEN HAS AN ANTIGENIC Properties but the HAPTEN PLUS
PROTIEN CARRIER IS IMMUNOGEN
Antigens are the a ligands that react with the products of an
immune response .
Hapten-carrier conjugates have native
antigenic determinants of the carrier as
well as new determinants of the hapten
Epitope ( - antigenic determinants ) :-
are the sites either (on or) within the antigen with which antibodies or T-cells
receptor reacts
paratope :- the sites on antibodies which react with the antigen .
epitope size ( small )
conformational linear
conformational
site are on antigen surface
or internal that expressed only when the
antigen has been partially degraded in
vivo
valency of antigen :- e.g multivalent
i.e the antigen molecule carry a number of different epitopes
( some times 2 or>)
some of which specify antibody A others specify antibody B .
valency = total no . of epitopes the antigen pocesses .
Antigenic determinants are usually
limited to those portions of the antigen
that are accessible to antibodies shown
in black for this iron-containing protein
EPITOPE (ANTIGENIC DETEREMNANT):-
The portion of Ag that binds specifically with
the binding site of Ab (paratope) or a receptor(s) on T_lymphocyte
SIZE CONFORMATIONAL STRUCTURE
The size and the structure of epitope are complementary to that of paratope
.i.e. they must have approximately the same dimensions
WITH RESPECT TO THEIR STRUCTURE ,A g MAY HAVE THE FOLLOWING
CHRACTES :-
Ag may have only a single epitope of a given specificity on its surface which is
capable to bind with antibodies , such Ag is called UNIVALENT AND UNIDETRMINANT
(one kind of specificity ) for example hapten
Ag may have two or more epitopes (which determine the specificity ), the A g in
this case is called MULTIVALENT (which determine the number).
If the epitopes are of the same type, the Ag is called also UNIDETERMINANT
(UNIDETERMINANT MULTIVALENT and if they are of different types called
MULTIDETERMINANT (specificity,
MULTIDETERMINANT- MULTIVALENT
).
UNIVALENT
UNIDETREMNANT
MULTIVALENT
UNIDETREMINANT
MULTIVALENT
MULTIDETRMENANT
In an antigen, the same antigenic determinant
repeated many times
T-dependent antigens are characterized by a few copies
of many different antigenic determinants
factors Affecting Immunogenicity .
Foreigness chemical complexity molecular
size
- : foreigness – A
the immunogenic substance must be forign to prduce immune response .
The greater the foreignness, the more will be the reponse
*- identical twins smaller or no response
*- brothers with the higher immune response
same tissues compatibility
the same blood groups .etc ………….
- B.CHEMICAL COMPLEXITY :
*- MOST of organic molecules are immunogenic expert lipids
*- proteins are the strongest immunogenic substance .
*-Polysaccharides most of them are haptens but they become complete Ag in
cases of
* peneumococcal polysaccharide .
* Lip polysaccharides in cell membrane of gram (– ve ) bacteria.
- Glycoprotein's : - *
Are immunogenic ex blood group Ags ( A,B,AB,O,RH )
- POLYPEPTIDES & nucleic acids : - *
Are weak immunogens
are not antigenic or immunogenic - : lipids - *
- : C.molecular size
usually the larger the molecule the stronger the Immunogenicety .
M.Wt below 5000 DA ARE NOT IMMUNOGENIC
MACRO MOLECULES are the most potent immunogens .
( e.g. albumin m.wt 40.000 Da
Globulin m.wt 160 kDa
Macrocyanin m.wt 1000 kDa
with molecular size . proportionally The valence of A.g increases
Macro molecules are easily to induce phagocytic ( as example ) and easier to
be phagocytosed
Quaternary structure are the most Immunogenic
The more complexity , the more Immunogenicety
Superantigens activate a large fraction of
T cells in contrast to conventional T-
dependent antigens
of determinant but many of such one kind MULTIVALENT since it has only
determinant on each molecule
Ex. Many poly saccharides & homo polymer (e-g peptide chain of the
some .A. Acids .)
are multi determinant & valent such molecules have many some antigens - *
epitopes of different kinds (multi specificity ) but only one of each kind ( mono
valent )
Ex. Most proteins .
chemically complexed compounds or polymerized proteins , High M.WT - *
(quaternary structure or heteropolymerized proteins are usually .
Ag ( multi specific) , multivalent Ag (more than one Multi determinant - *
epitope of each kind)
) What kind &How many of such kind )

Antibody binding site ( Paratope ).
Binding of Ag & Ab
Affinity :- the strength of attraction and binding between an epitope( mono
valent ) of an Ag and the antigen combining site of Ab molecule ( Paratope
) .
Avidity :- The strength with which ( multivalcnt ) Ag bind to its
antibodies ( Abs).
This depends on the affinities of the individual
combining sites of the determinants
on the antigen
( chemical complexity )
Electrophoretic separation of serum proteins
ANTIBODIES and their STRUCTURES
*Classes of antibodies .
IgM , IgG , IgE , IgA & IgD .
A = COMPLEMENT BINDING
SITE
B = NEUTROPHILS & MACRO- PHAGE
BINDING SITE
VARIBLE = ANTIGEN BINDING
SITE .
C
o
n
s
t
a
n
t

B
C
o
n
s
t
a
n
t

A
C
o
n
s
t
a
n
t

A
C
o
n
s
t
a
n
t

B
H
e
a
v
y


c
h
a
i
n
ANTIBODY STRUCTUR
CH3
CH2
CH1
Hinge bonds
The basic structure of immunoglobulins
Rotating antibody
ANTI BODIES
POLYCLONAL ANTI BODIES
- INDUCED AGAINST WHOLE ANTIGEN .
- LESS SPECIFIC
- PRESENT IN SERUM
MONOCLONAL ANTIBODIES
INDUCED AGAINST ONE EPITOPE .
(I.E . SMALL PART OF ANTIGEN )
- MORE SPECIFIC .
- PRODUCCED BY HYBRIDOMA
TECHNOLOGY .
EPITOPE
EPITOPE
POLYCLONAL Ab.
INFECTION
MONOCLONAL Ab MAb MAb MAb
i-Immunization
antigen
HybrIdoma technique
+
fusion
B-CELLS
MYELOMA
CELLS
HYPRID CELLS
Tissue culture
supernatant
Fluid
Ascetic fluid
selection of
Desired
Clone
+ MICE
II_ FUSION
TUMOR MICE MICE
Immunoglobulins
"Humoral antibodies”
They are formed of two identical units each of them is formed of :-
A) heavy chain B) light chain C) hing region
A) Light chain 2ì( lambda)
but never 1ì and 1K
K ( Kappa )
2
B) Heavy chains :
* M-Wt 53.000 - 75.000 Da
*- heavy chains are hold together with (disulphide bonds) .
*- Fixed region contain 2k or 2 ì .
*- The variable region contain a mixture of K,ì.
*- both L& H chains contains the following region :
Light chain contain variable (VL) and hyper variable (VH) regions
Heavy chain contain variable and hyper variable regions.
VARIABLE REGION CONSTANT REGION
* Amino terminal * carboxyl terminal
* The amino acids differ * A. As are similar in differentì
on ì to another specificity. * it contain the effectors domain which
is responsible for the
* The VL & VH are adjacent to initiation of the process
each other forming paratope . by which the body gets-rid of Ag. .
* They have sub-regions of the
variable region (hypervariable)
-
-
-.

-
( hyper variable )
It is responsible for
Designation of Ab class & its
distribution.
These regions have extreme
variability in their A .As
sequence in different antibodies
and they are responsible for
binding with Ag(s)
{ CDRs}
comptementary
detemining
regions
C) Hinge region :-
* CH 1, CH 2 , CH 3 : occupies ¾ that of Heavy chains the other ¼ is
VH .
* The Hinge region lies between CH 1 & CH 2 .
* It is flexible & allows movement between the two antibody binding
sites .
* The hinge region is digested by protease (e.g. pa pain ) which splits it
into :-
( i ) antigen binding fragments (fab) = They are 2 identical fragments containing the
antigen binding site .
( ii ) crystallization fragment (FC ):-It contains the effectors )
Structures and function
Of
Specific Immunoglabulins
*- Ig(s) are glycoprotein's in the gamma globulin fraction of serum proteins (albumin ,
fibrinogen , globulins (o , | and o ) .
*- they are produced by B- lymphocytes or plasma cells in response on
immunogen (or Ag ).
General Ig structure :-
*- 4 poly peptide chains.
*- they are linked covalently by disulphide bonds
*- the 4 chains , monomeric Ig structure ,are
composed of 2 identical heavy poly peptide chains (H)
2 identical light poly peptide chains (L)
*- Heavy and light chains :
*- H- chain :
*- Have a M.wt of 50-75 KD (~Twice that of L chain )
*- H chains contain ~ 400 A.As (~Twice that of L chain )
*-A. As differences in the .COOH terminal portion of the heavy chain (CH) identify 5
distinct H-chains isotypes .
* Each H chain has 4 or 5 domains :
1 domain in the variable and 3 or 4 in the constant
3 IgG ( ¸ ), IgA(· )&IgD( o) Or 4 Igm ( u)&IgE( c )
Total

= 1 Variable + 3 constant or = 4constant 1 + Variable
The Hinge region
*- It is the portion of the H-chain between CH 1& CH 2.
*- there is no homology between it and the other H- chain domains, thus .its
sequence is unique (sole) for each Ig type and subclass
Notes (1) -Each L- chain has 2 domains 1 VL
1 CL
(2)- Folding of the polypeptides chains brings the hyper
variable regions of the VH and VL domains into close
proximity .
(3)- this folding creates a 3-dimensional structure that is
complementary to the epitope (last figure )
IgM & IgE do not possess a hinge region but have one more CH domain.
These structure explain why both IgM & IgE have 4 domains on the CH chains but
not like the other types (which have only 3 domains on CH)
*-In this region (hinge), inter chain disulphide bonds forms between the arms of the
Fab fragments preventing them from folding and therefore
, rendering this portion of the molecule highly susceptible to fragmentation by
enzymatic attach .
* -The hinge region is highly flexible and allows for movement of the Fab arms in
relation to each other .This motility explain why native antibody
molecule do not activate complement , whereas those in an immune
complex do .This is because , the native Ab is not in the appropriate
configuration t1/2 or half life of( Abs) .
*- These heavy chain isotypes form the basis of the 5 Class of Immunoglabulins
molecule IgG (¸) ,IgA (· ) ,IgM ( u),IgD ( o)and IgE (c ).
*- H chains Classes ¸ and · are subdivided into subclasses of molecules
¸ ¸1 , ¸2 , ¸3 , and ¸4
And · · 1 and · 2
The subdivision is based on the greater similarity of A.As sequence shown by
subclasses of the same class
i.e. ¸1 , ¸2 , ¸3 etc,. Than is shown by different classes
(i.e. o , ¸ , or u)
*- The heavy chain subclasses determine immunoglobulin subclasses
e.g. ¸1 = IgG1
¸2 = IgG2 , ¸3 = IgG3 etc,.
*-L-chains :-
* Are composed of ~ 200 A. As .
* They are of 2 types ( K= Kappa or ì = lambda ) .
{ based on their structural (antigenic) differences }
* All Igs classes have 2K or 2ì chains but not kì or ì k .
ex. * The proportion of K/ì = 3/2 (human Ig) .
ì- chain Isotypes :-
*- There is no isotypic variations in K chains
*- There are 4 distinct ì chains 4 different isotypes .
*- All the 4 subclasses are present in each of the Ig classes
i.e. in IgM , IgE , IgD etc.
*- Disulphide bonds Hold together the 4 polypeptide chains
in Ig molecules .
* - There are 2 types of disulphide bonds : -
H – L chain
L – L chains
H – H chains
1- Inter- chain disulphide bonds :
Single L-L bond only in
Hinge But also in Ig A2M (1)
region COOH-terminal such bond can
of the H chain occurs in all Ig(s)except occur under path-
Ig A2M (1) which ogenic conditions.
Lacks an inter chain (e.g. Bence Jones
They can be 1:15 depending disulphide bond protein ) seen in
On the class & subclass types urine of some
patients with
multiple myeloma
occur between
INTRA CHAIN DISULPHIDE BONDS :
*- occurs within an individual chain .
*- they are stronger than inter chain bonds .
*- they no. of intra-chain disulphide bonds varies depending only on the number of
domains in the molecular .
Light chain have 2 intra-chain bonds .
*- human IgG, IgA, IgD heavy chains have 4 intra_chain bonds
*- human IgM , IgE heavy chains have 5 intra-chain bonds .
*- Each H& L-Chain has a variable (v) and constant (c) region
*- V region lies in the – NH2 terminal portion of the molecule .
*- The V region has a wide variation in it’s A.A composition .
*- The C region lies in the - COOH terminal end of the molecule .
*- The C region has a much more constant A.A Sequence except for minor inherited
changes
*- The variable regions associate with appropiate constant regions .
so that a variable H – Chain regions (VH) does not occur in an variable L – Chain
(VL) and Vise versa .
*- However , a particular VH chain sequence may occur in more than one
H – Chain class ( i.e IgG, IgM , IgD ,IgA and IgE ) .
*- Thus during class switching in an immune response e.g when B – cells change
their production from IgM to IgG heavy chain
only the constant regions of the H (CH) changes and the antibody specificity
remains the same .
HYPER variable regions
*- they are particular areas within the variable regions
That are highly variable in A. As sequence .
*- THESE hyper variable regions often called complentary determining regions
*- THESE regions occurs at simillar A.A positions in an relatively invariant
molecules .
CDRs :- they are short polypeptide segments lining near A. As positions 30,50
AND 90 in the variable regions of both L and H chains .
Note :- the variability range ( index ) used is an arbitrary scales of the no. of
different A.AS found in each position if 100 different Light chain were analyzed .
*- the hyper variable regions are important in the structure of the Ag binding site
( paratope ) .
*- L – chain have 3 hyper variable regions ( the last figure )
*- H – chain have 4 hyper variable regions although,
ONLY 3 OF THE 4 have been associated with epitope recognition
*- each Ig chain consists of a series of globular regions or domains enclosed by
disulphide bonds ( intra or inter ) ?? Chain disulphide bond .
*- The A.AS sequence of the domains show a high degree of homology
( i.e the sequences are very similar ) .
24-34 50-56
89-97

variability
FR1 FR2 FR3
FR4
CDR1 CDR2 CDR3
NO OF AgS
CDRS
FRs
variable region
Structure of the variable region framework regions
- Properties of Ig :
IgE IgD IgM IgA IgG
c
-
190
11
0.05
2
-
-
?
+
o
-
180
13
3
3
-
-
-
-
u
-
900
12
120
10
++++
-
+
-
o
o1 o2
160-400
7
200
6
-
+
+++
-
H – CHAIN ¸
H – CHAIN SUBCLASS ¸1, ¸2
M.Wt 150
Carbohydrate (%) 3
Serum conc(mg %) 1200
Seru t ½ ) days( 21
- : Functions
Complement activation ++
Opsonization ++++
Antiviral activation ++
Mast cell sensitization -
% of their M.WT ) 13 - 3 ( - : Immunoglobulin are glycoproteins
OLIGOSACCHARIGES + PROTEIN
*- THESE oligosaccharides are present in CH
2
or CH
3 .
*- N -glycosidic bonds usually link N- acetylglucosamine in the carbohydrate moiety
to asparagine residue in the peptide c-chain of Ab
[ linkage with the enzyme N -acetylglucosamine .
- Asparagine transglycosylase ] transferase
*- t ½ of Abs in the circulation depends on the status of oligosaccharide side chain
*- the oligosaccharide side chain of Ab terminate with galactose to which sialic acid
is bind .
*- when Abs have the sialic acid removed by the enzyme neuraminidase , they
become susceptible to degradation in the liver .
*- in this case the terminal galactase bind to a receptor on hepatocytes and the
entire molecule is , then , interenalized to the cell for degradation via
Proteolytic enzymes in lysosomes of the cells .
Immunoglobulin fragments: Structure/function relationships
Digestion of Abs with Restriction enzymes (Immunoglobulin fragmentation) as
well as Structure/function relationships:
S-S
Ab
F(ab)2 FC
S-S
Restriction enzymes digestion of Abs :
1) Papain : digest above hinge region so it leaves 2 Fab fragments each is monovalent
And crystalline fragment (FC)
2)Pepsin: digest away most of FC
Fragments below the Interchain disulphide bond
(below the hinge region) it give one large fragmentsF(AB)2 which is consist of two Fab
fragments joined by the disulphide bond
Thus , it is bivalent ,possessing the ability to bind and form agglutination
papain
Fab FC
Monovalent
S-S
Ab
F(ab)2 FC
S-S
Figure 4 Immunoglobulin fragments:
Structure/function relationships
Classes of antibodies
They are 5 isotypes
The class of Ab depends on the A.A: sequence
of the constant regions of the heavy chain .
- Immunoglobulin M (IgM) : - *
* it is a pentamer ( 5 molecules ) .
* they are linked together by disulphide bridges at the COOH terminal end
of the heavy chains as well as an additional poly peptide chain ( joining chain)
* this type of Ab account for 8-10% of the total PLASMA ANTIBODIES .
* it is the most abundant Ab produced by the faetus .
* it binds with viruses and bacteria
- Immunoglobulin g ( IgG ) : - *
* it is a monomer
* it accounts of ~ 75 % of the total antibodies .
* it is important for elicit ting the immune response to Ags
* it is only antibody which pass through the placenta to protect the faetus.
IgM
*-
immunoglobulin D ( IgD):-
* It is a monomer ACCOUNTING FOR < 1% & TOTAL ANTIBODIS .
* Its function is controversial .
*- immunoglobulin E ( IgE):-
* It is a monomer ( below 0.004 % & the total Abs)
* It is present in spleen , tonsils , mucus membrane of lungs GI
* On binding with ag it releases histamine from mast cells leading to
hypersensitivity .
* It provides immunity to intestinal parasites .
*- immunoglobulin A ( IgA):-
* MONOMER , DIMER or TRIMER( mostly dimer )
* Like IgM the units are linked by disulphide and j chain
* it is found in tears , saliva , intestinal treat secretions
* it binds with Ag preventing them from tissue adherence , colonization ,and
making them more phagocytosed .
Laboratory Methods
Serology
In vitro Ag & Ab reactions called serology
It provide methods for
i) Identification (Diagnosing) ( ii ) quantization of titre of Ab (and \ or) Ag
Titre : or the level of Ab (s) in the serum can be measured by using known Ag
The titre may have diagnostic
prognostic
Ex. A rise in Ab titre between acute &convalescent serum can be used as a
diagnostic tool for a specific disease
The titre is defined as the greatest dilution of serum (which contain the Ab under
consideration ) that reacts which the antigen ( i.e. gives +ve result ) .
or
- the forces involved in Ag-Ab reactions are greatly affected by various
environmental factors :-
*- The Ag- Ab complex is not bound firmly together .
*_This complex may even dissociate spontaneously .
* physiologic ph & salt concentration promote optimal union of them .
*- the force of attraction tend to be weaker in
a) very acidic .e.g. 0.01M
b) very alkaline medium
i.e pH 4 and alkaline ( i.e. above pH 10 )
- temperature :- it plays an important role :
* the higher the temp ( up to 50 – 55
0
c ) , the more rapid is the rate of reaction
between Ag & Ab .
* the reason is the increase in kinetic motions of the reactants ( Ag & Ab )
various forces act to hold the Ag-Ab complex together :-
* The maximum attractive forces stabilizing Ag-Ab complexes
Are van der weal forces
Ionic bonds
1- van der weal forces :-
* occurs because of spatial fit ( the below fig )
* these forces of attraction hold Ag to Ab only
When the two molecules have complementary shapes (a)
2 Epitope
puratope 2
(a) (i) significant changes
In the shape of epitope 2
Into 2a
puratope 2
a 2 Epitope
( b )
these change precludes its ( 2a ) interactions with the matching binding site of the
original Ab .
* When the molecules have less similar shapes ( b) , these forces are less effective
(b)
2-Ionic bonds :-
* They are patterns of complementary electric charges on the molecule .
* The electrostatic interactions tend to hold the molecules together .
COO
COO
COO
Affinity :- the strength of attraction between a single epitope and its matching
paratope is the referred to as the affinity of the reaction between the two reactants .
Ag-Ab complex of low affinity dissociate readily
Avidity :-
* It is a related term to affinity
* It refers to the strength of the interactions between multivalent antigens and the
population of Abs that they have included .
NH3
+
NH3
+NH3
*- Avidity is influenced by the affinity of individual Abs for their
(A) epitope
(B) the valency of Ag and
(C) the valency of Ab
tertiary structure of protein :
*- the ability of Ab to bind with Ag can be affected by altering the tertiary
structure of any of them
ex.
insulin which is composed of A&B chains Ab to either one of these chains can
be produced by
(a) splitting the chains
(b) purifying tem
.e.g. a pig) ) (b) injecting
them into foreign host
the pig will produces Ab to the particular chain that was injected
*- if the host (pig) Abs are injected back into the animal species that supplied the
original insulin (man) , the abs will not react with intact insulin molecules .
*- This is because the tertiary structure of native insulin is such that the
epitopes on the A & B chains are not accessible ..
Now , it is generally accepted that in a given poly peptide
the A .As that are spatially accessible because of
Tertiary structure of this protein are only immune
reactive
Large enough to be visible
with naked eye
*- The physical state of the antigen is responsible for the identification of Ag –Ab
reactions and the naming of Abs .
*- The same Ab molecule could , in fact , be described by each of the following
terms :
(1) Agglutinins are Abs that aggregate cellular Ags.
(2) Lysins are abs that cause dissolution of cell membrane .
(3) Precipitins are abs that form precipitate with soluble Ags .
(4) Antitoxins are abs that neutralize toxins .
procedures must be involving direct demonstration and observation of
reaction ..
The relative sensitivities of the tests for Ags and Abs are
Presented in table 8.1 page 156 [ immunology , 3
rd
edn ].
A- Agglutination Reactions :-
cellular Ags and identify Agglutinins Serve to detect and quantities
Bacterial cell white blood cells red blood cells .
**-- when the cells intact with the appropriate Ab , they clump together and eventually

b a
form masses
*- When Ab agglutinates bacteria in the body opsonization occur .
*- Agglutination occurs because Abs and at least bivalent .
*- Two sites on the Ab and multiple sites on the Ag
Ag – Ab lattice formation

that can
build up into increasingly larges coupled
lattice structure
Example widal test :- (diagnostic test of typhoid )
*-Ab of patient serum is measured by adding a constant mount Ag
(e.g. salmonella typhi ) to serially diluted serum .
*- After incubation , the test tubes are examined for visible agglutination .
*- the last tube (i.e the highest dilation of serum ) showing agglutination
is referred as the titre .
- lyses Reactions : - B
In the presence of a complement an Ag – Ab reaction , on a cell
membrane , may result in membrane damage that leads to cell lyses
This phenomenon is important in the host's defense against condition
such as microbial infection or cancer ( graft cell , virus infected cells
, etc…………….(
- : Haemolysis - *i)
In which the Hemoglobin is released from R.B.C, is a requisite
phenomenon for the complement fixation test .
- bacteriolysis : -- *ii)
cells of gram (– ve) bacteria are undergoes immune lyses under certain
condition .
- : cytolysis -- *iii)
involves the destruction of other cells types (e.g. lymphocytes ).
- : precipitation - C
* occurs when the Ag is soluble instead of cellular
*therefore a large number of molecules are required for lattice formation and a
large no .of lattice must be formed for an aggregate to be formed and visibly seen .
*when soluble Ag (s) come intact with specific Ab. They aggregate
(i.e precipitate )
Three conditions are present
A- where the (Ag) is very low with excess Ab (zone of Ab excess ),
Formation of complex occurs
But
Residual Ab remains in the supernatant
B- As more Ag is added , large aggregate is formed
In the (zone of equivalence) ,
maximal Ag-Ab complex are formed and precipitated
C- Instead of reaching a plateau , this curve comes back down to zero
with increasing the mount of Ag (zone of Ag excess )
* this is because the lattice size becomes too small to precipitate .
* In extreme Ag excess . the complex will be trimmer
i.e one Ab +2Ag
Note:- the soluble Immune complex are not processed efficiently by
the reticuloendothelial system ,and ,this cause damage (how??)
INDIRECT HCG :
Examples 1 :- determination( and\ or) detection of HCG by using indirect methods .
(i) an Ag will be added ( HCG ) .(from the kit)
(ii) Urine will be added ( excess Ag ) from a female may be pregnant .
(iii) Ab to HCG will be added
In case of positive In case of negative pregnancy
A state of Ag excess a state of equivalence will be reached
Therefore, no precipitation therefore, precipitation occur
Direct HCG assay :
* (i) Ab to HCG will be added (from the kit)
* (ii) Ag ( HCG of the test sample will be added) .
If precipitation occur ( positive) if, no precipitation occur ( negative )
A
m
o
u
n
t

o
f

p
r
e
c
i
p
i
t
a
t
e

Zone
Of
equivalence
Effects of increasing amounts of Ag on the total immune precipitate
obtained from a mixture of soluble Ag and its homologous Ab
Hyaluronic acid (HA) assay using excess
HA binding protein (HABP) :-
* HABP will be added in excess ( known excess ) (ACT AS Ab)
* Sample will be added ( containing HA) (ACT AS Ag)
* [ A state of Ab excess no ppt ]
* An radiolabelled HA will be added ( Ag )
Thus, precipitation occur ( IF +ve sample) and immune complex will be separated
and quantities by radio- immune assay technique ,in case of no precipitation, the
sample is negative
Immune diffusion
* It used for quantization of Ag (s)
* Thus, precipitate will also be demonstrated .
* If an Ag – Ab reaction takes place in semisolid medium (e.g. agar ) , band of
precipitate will be formed .
* The reason of precipitation , is the diffusion of the components (Ag & Ab ) towards
each other .
* A useful example is a double immune diffusion technique :-
Procedure :-
*
Ag & Ab preparations are placed in separate wells that are cut into a thin layer of
agar in a Petri dish .
* The reactants diffuse towards each other through the agar until they meet an
optimal proportions [ zone of equivalence ] and forms
( ppt ) bands
The advantages of the procedure is that antigenic relationship
can be detected by the precipitation pattern (s)
Ab
Ag
Ag
Fig (a)
Solid
Chevron
PPT
Zone of
equivalence
3 basic patterns are given :
(a)- in reaction of identity , the 2 Ags are similar , they will diffuse at the same rate
and the two precipitations bonds merges into a solid chevron ( fig b)
2- in reactions of non-identity , the two Ags are completely different and the lines
of the precipitate cross (fig c)
3- reaction of partial identity :-
* It is indicated by spur formation indicating that one of the
Ag(s) is cross-reactive ( but not identical ) to the other one .
* The spur occurs because one the Abs (b) does not react with the cross-reacting
Ag (Ag ac) but migrate past that Ag (Ag ac ) until it reaches an Ag (Ag ab ) that
Aga Aga
Ab
Fig b
Fig c
Aba
Abb
Aga Agb
Ayab Agac
Abb
Aba
Has an epitope for which it has specificity .
B- quantitative radial immune diffusion
* It is used routinely to quantities Ab in serum .
* For this purpose , an agar coated slide is used .
* The agar being impregnated by anti sera ( antibody to human IgG )
* SERUM samples are placed in wells in the sugar .
* As it diffuse through the agar and encounters the Ab, the IgG in the sample form a
concentric ring or halo precipitate .
* The diameter of the halo of precipitate directly correlate with the [ IgG] in the
sample .
Thus , the levels of IgG in the sample can be determined by referring a standard
curve based on halo diameter (s) of known concentration (s) of IgG
C-immune electrophoresis :-
* It was developed because the double immune diffusion technique.
(i) Could not resolve high complex mixtures of Ags .
(ii) and, a more sophisticated technique was needed .
In this procedure :
(a) Ag is placed in wells in agar on a glass slide and then , subjected to
electrophoresis through application of an electric current .
(b) Under these conditions , the individual Ags or antigenic components ( in
the same sample ) migrate through the agar at variable rates .
(c) If Ab is placed in a well that runs the length of the slide parallel to the path
of migration , the reactants will diffuse towards one another and form separate arcs
of precipitate for each antigenic determinant
D- counter-immune ectrophoresis (CIE)
This technique Is
the double diffusion method + an electric current
Which plays as the migratory force which:
(i) – amplify the speed of reaction ( 24 hrs to 30 min )
(ii) Intensifies the precipitation bonds .
(iii) Increasing the sensitivity of the assay about 10 fold .
Procedure :-
(i) Ag & Ab are placed in wells and the current is applied .
(ii) in suitable buffer ( eg ph 8.6 ) the negativity charged Ags migrate
towards the anode , whereas the Ab [ which has no sufficient net charge ] migrate in
the opposite or counter-direction , as a result of endosmosis .
• Precipitation occurs where the reactants melt .
D- Antitoxin :-
* If a serum contain an antitoxin ( i.e. antibody to a toxin ) , the Ab . Will neutralize
the toxin examples :-
- Suppose serum containing antitoxin is mixed with toxin ( in vitro ).
- Then , after a few minutes , a small amount of the mixture is injected into an
experimental animal ( in vivo ) .
- The animal will be protected against the introduced toxin , and thus , its deleterious
affects disappear because of antitoxin is present .
the virus haemagglutinate R.BCs) ( - : Clinical example
* To examine the serum of a patient suspected of having influenza ,
*1) The patient serum is mixed with known influenza
2)add red blood cells
i- if Ab is present haemagglutination will be prevented
.i.e the sample is positive
this is due to the ability of Ab to bind with the virus and block its ability to
haemagglutinate the R.B.Cs
ii- if no Ab is present , haemagglutinate will occur .
virus + R.B.Cs haemagglutination occur
.i.e the sample is negative
E-Flacculation :-
it is another form of Ag –Ab reaction that
occurs if the Ag is neither cellular
nor soluble
but it is an insoluble particulate
The venereal disease research laboratory (VDRL) test is a slide flocculation test
used for the diagnosis of syphilis .
• The VDRL make use of heterogenetic ( heterophillic ) antigen shared between the
Spirochete of syphilis & normal beef heart .
* The Ag used is a water insoluble cardiolipin that had coated the surface of
cholesterol particles that were added to the system .
* These form visible aggregate indicate to the presence of Ab ( reagin ) in the serum
of patient for syphilis
[ reagin is Ab type which flocculate (or ppt) an Ag that is neither cellular nor soluble
but it is insoluble ]
* The test can be performed on a glass slide .
VDRL TEST FOR Syphilis
Technique :-
cholesterol particles + normal beef heart extract
( inert support ) ( antigen like substance )
Insoluble antigen
serum
( A.Bsource )
visible aggregate
Which can either seen by
The naked eye
Using a microscope
and green filter
Ag-Ab reactions in which the visible manifestation requires Participation of:
a) Accessory factors
b) Indicators system
c) Specialized equipment
COMPLEX SEROLOGICAL PROCEDURES
- fluorescent dyes : - A
e.g fluorescein isothiocyanate ( FITC)
* FITC can be conjugated to Ab. Molecules to visualize of the molecule under (uv) or
a fluorescence microscope .
* such labeled Ab. May then be used to identify Ag(s)
(i) Direct immunofluorescence assay :-
* The method uses Ab. That is specific for a particular Ag
parasite
* This Ab is labeled with a fluorescent dye (FITC)
* This conjugate is allowed to react with unknown tissue or organism .
* IF the Ab reacts ( i.e +VE the result ) , it will visualized as green stain on the
fluorescence microscope specimen when it examined under the
using uv light
(b)
or
b
y
Examples :-
Identification of Trepenoma palladium ( syphilis ) in an extracted from a patient
suspected of having syphilis .
- Procedure :-
*1) The slide is coated with the Ag .
*2) Ab tagged with FITC is added .
* 3)Excess Ab is then washed .
* 4)Then , the slide is examined with uv fluorescent microscope .
* Trepenoma palladium is fluoresce against the black back ground
this methods can be extended for other pathogens .
(ii)-immune peroxides technique :
If viral antigen in tissues will be detected, horse radish peroxides
is conjugated with the Ab .
*-(1) After the enzyme – Ab complex has reacted with the tissue (Ag) .
*- (2) Excess Ab is washed .
*-(3) And , an appropriate enzyme substrate is added to the tissue
section .
*- the bound Ab. Is detected by the presence of a dark precipitate
*- Advantage of immune peroxides technique over the immune fluorescent
technique:-
*- The specimen can be stained with conventional Histochemical dyes
So structural details can be seen ( noted )
. light microscope the tissue con be examined by standard - *
(iii)- Indirect immune fluoresce technique:-
*- The procedure use Ab ( secondary ) ( against ) another Ab
( primary ) of patient .

*- the primary Ab is the patient' serum detection of Abs.
*- the secondary Ab is covalently conjugated whit fluorescent
compound (FITC)

antihuman( INJECTED Ab in host is FROM rabbit Of secondary Ab is ex. - * - *
gamma globulin anti sera . HUMAN )
.i.e Produced against Ab used to immunize rabbit and that Ab
which will be examined latter on (unknown conc.)
Technique :- (this procedure allows for detection of Abs,)
Example serodiagnosis of syphilis by the fluorescent trepenomal antibody
absorption (FTA-Abs) test
*-(i) -T. pallidum is fixed to a slide
*-(ii)- the slide is flooded with the patient serum (staining Ab )
*-(iii)-If Ab to spirochete are present, the Ab will reacts (bind) with the
organism on the slide
* (iv)-Excess Ab (serum) must be removed with washing , to detect the bound Ab
only .
*-(v) the Ag-Ab complex formed is them treated with the fluorescein – tagged Ab to
human gamma globulin, the excess Ab is washed carefully.
*-If the patient's serum contains Ab (+ v e) against the T. pallidum, fluorescein
organism will be seen when the slide is examined with fluorescence microscope

**-- Ab fluorescence
conjugate is
binded .
(uv ) is used Microscope
Indirect Immune fluorescence assay is also used for -
- Detection of Antinuclear Antibodies (Ana) :
( e.g. DNA , RNA & His tone)
ANA are present in systemic lupus erythroMatosis ( SLE ) , some
Times in rheumatoid arthritis and other autoimmune collagen –
Vascular diseases .
- Example (SLE ) :
* The procedure is similes to that of T . palladium .
* The Ag is ( DNA ) histone in form such as
Animal Buffy coat calls
Human Buffy coat calls
Rat kidney section
beef thymus
Lymphoid Thymus
Lymphoid organs
FITC
Patient’s
Serum is
added
Slide coated
with ag in
(excess)
- : Haem agglutination Inhibition teat
It involves The agglutination of R.B.cs by
(haemagglutinin(s))
Ab(s)
Certain virus
particles (influenza)
Or
other
substances
It demonstrates the presence of serum Ab to haemagglutinating viral substance .



- Technique :
R.B.C s
serum sample Ab from the
Which contain Ab kit that make
Prevent haemagglutination haemagglutination
Agglutination occur no agglutination
The sample is negative the sample is positive
Similar test can be used to detect soluble Ag(s) which able to
react with and neutralize a haemagglutinating Ab .





R.B.Cs from kit


Abs from kit which
Capable of haemagglutination
serum Ag which
Prevent haemagglutination
haemagglutination
Inhibition
Sample is positive
haemagglutination
takesplace
Sample is negative
- passive agglutination:-
in the conversion of a reaction system from one that
precipitate one that agglutinate
Thus yields a more sensitive indication of
the presence of antibodies .
The detectable antibody is called rheumatoid factor
- RHEUMATOID ARTHRITIS Example :
The use of latex particles in the diagnosis of rheumatoid arthritis (soluble Antibodies
) is an example of passive agglutination .
Principle :-
In this disease ,the patient produces an Ab (Mainly IgM) to his own IgG
Technique :-
*-(i) latex particles were coated with IgG.
*-(ii) patient 's serum is added (which contains antibodies IgM)
*-(iii) Agglutination indicates the presence of Antibodies (Ig?)
(i.e The test is positive )
Bis-diazotized diphenyl :-
it is a coupling reagent that can be used to
proteins
conjugate : or R.B.Cs
Haptens
and thus
Passive haemagglutination
Occur.
to
Thus :
*- Addition of serum containing Antibodies to these substances (proteins or
haptens ) allow the detection of these specific antibodies to these substance by a
technique called Passive Haemagglutination
rose –waaler Test :
which detect rheumatoid factor in serum of the patients suspected to
have an anti-IgG auto-antibody .i.e. .(IgM to an accumulated IgG
- Tannic acid –treated a sheep R.B.Cs (S R.B.Cs) are coated with rabbit IgG
Antibodies specific for these S. R.B.Cs

















haemagglutination
the sample is positive IgM
from
the kit
serum from a patient suspected
to have autoantibodies R.B.Cs
Tannic acid
coated by IgG
Coomb's (antiglobulin) Test :-
* In certain people .Abs directed against antigenic determinants (e.g. R.B.Cs
antigens )are able to form visible aggregates when subjected to :
1- precipitation 2- Agglutination
*-To demonstrate the presence of Abs in such cases the coomb's (antiglobulin)test
may be used .
*- The test involves the addition of Ab direction against gamma globulin : which
provides a bridge between two antibodies coated call or particle
Thus ,The major use of the coomb's test is to detect the Non agglutinating
(haemagglutinating )anti-red blood Cs Abs.
(1) – Direct coomb's test
* It is used to detect call bound antibodies
Technique :-
You must use EDTA BLOOD then centrifugation
*-(1) The red blood calls (bound antibodies )are washed free from serum and the
unbound antibodies (to be leaving the bound ones ).
* - (2) Antiglobulin serum is added directly to this call suspension




*- The direct coomb's test is of value in the detection of antibodies to
R.B.Cs associated with hemolytic disease of new born (e.g.
erythroblastosis fetalis ) and auto immune anemia or disease .
*- The Abs associated with these diseases have the ability to attach to but
not agglutinate the target R.B.Cs .
*- These absorbed Abs can be detected by the use of Ab (i.e. coomb's Kit
serum ) to This human gamma globulin .
(2) indirect coomb's test :-
*- It is used to detect the presence of Circulating Antibodies.
*- It is of value in detecting IgG - associated antibodies in the serum of
woman who is though to be (a) sensitized to Rh antigen And
(b) at risk for carrying an erythoblastbotic febus .
Technique :-
*(1)- Serum sample (containing Ab ) is incubated with donor R.B.Cs( contain
Rh antigen).
*- (2) Then ,the cells R.B.Cs are washed off ( to remove excess Ab ) .
( (3) The anti globulin (coomb reagent) reagent is added ( kit -
*
Faetus like blood
Serum
serum

Anti globulin commb reagent




Ab is absorbed haemagglutination ( + ve ).
No Ab .(No absorption ) haemagglutination ( - ve ).
Viral Neutralization :-
It is very similar to haemagglutination Inhibition on ( i.e. it is a
neutralization event ) .
*- Principle :-
The assays is based on the ability of specific Abs to interfere with
Some biological function of the virus under consideration ( usually
The infective property is blacked ) .
( 1 )- Cyotpathic effect ( CPE ) :--
certain virus + cells ( in tissue culture ) cell destruction .
2 )- the CPE is useful in the search for virus neutralizing Abs in serum sample.
- Technique :
- *
**_ serum suspected of containing Ab is added to a virus suspension .
**_ Susceptible cell culture is inoculated with the mixture .
If the culture fail to develop
CPE (no killing ) ( + ve )
CPE developed (killing )
no Neutralizations
( culture Cell death ) ( - ve )
– Radio immune assay (RIA ):-
* - It is and extremely sensitive method for quantization
of any substance that is Immunogenic or heptenic and can be
labeled with radioactive isotope e .g. I (25I) .
Liquid phase RIA :-
It depends on the competition between labeled (Known ) and
Unlabeled ( unknown )antigen for the same antibody .
Abs are interfere with the
Ability of the virus to kill
Tissue culture cells
Unlabled Ag[unknown amount] Labeled Ag (known amount)
Ab specific known amount
The radioactivity of
either
Is then determined
( i ) Separation of this complex by immunological method by
secondary Ab or by Precipitating agent . [ (NH4)(SO4)]
ii) Separation of this complex by physiological methods
(a) centrifugation
(b) Decantation .
PPT (commonly used)
The supernatant
.
A Calibration curve based on using serial dilution of known
unlabeled standard ( instead of the serum ) is used for
calculating of un known samples
C
P
M
conc.
Immune reaction product
(Immune complex)
*- Solid phase RIA :-
Liquid phase is modified by :-
( i ) adsorption or covalently linkage of Ab to solid matrix ( solid phase
RIA) .
( ii ) The unlabeled Ag (sample ) is added followed by the labeled one
( Antigen or Antibody ) .
Then
The bound versus free Ag can be determined by
Using reference calibration curve ( as before)
[ washing steps]
Enzyme linked Immune Sorbet Assays { ELISA } :-
*- ELISA is both highly sensitive ( > 99% ) and specific ( > 99% in
high – risk populations ) .
*- It can be for the assay of either Ag(s) or Ab(s) .
*- Ag or Ab can be attached to solid phase support ( plastic
surfaces , paper disks ) and still retains its immunologic activity.
*- Either Ag or Ab can linked with an enzyme e.g. ( horse
reddish peroxidease alkaline phosphates ) .
*- substrate is added and the color absorbed by the enzymatic
procluct is then quantization and compared with a calibration
curve .
Example :- detection of Ab(s) to the human HIV :-
*- The virus is grown in vitro in a human T-cell culture .
*- purified whole virus is disrupted 8 viral proteins are immobilized
onto plastic beads or multi well trays .
*- Abs to any of these antigens will bind with them & immobilized .
*- Excess proteins are removed by washing the beads ( or wells ) and
an enzyme linked anti human gamma globulin antibody is added .
*- The presence of this second Ab can be detected calorimetrically
by adding a substrate for the enzyme that will yield a colored end
product
.
*- The rate of substrate degradation is determined by the amount of
enzyme – labeled Ab that is bound which is proportional with the
amount of Ag in the solution being tested.
*- the color change can be measured quantitatively in a
spectrophotometer .
* Double Antibody sandwich ELISA :-
It is used for the assay of Ag ( e.g. HBSAg ) uses tow Abs as
below :-
( i ) first Ab ( specific e.g. HBs Ag ) is coated on a plastic surface
( poly styrene), the solution being tested for HBs Ag is then
applied to the surface .
( ii ) Washing of any un reacted material .
( iii ) The second Ab ( ie enzyme linked anti HBs Ag specific Ab is
then applied .
( iv ) Any excess conjugate is rewove by washing .
(v) finally substrate is added to the detest the present of En2

ABO group & Transfusion Reactions
*- ALL human erythrocytes contain all antigens ( i.e. Antigens that vary
among individual members of a species ) of the ABO group .
*- This is important system , which . is the basis for blood typing &
transfusions .
*- The A & B antigens are carbohydrates that differ by a single sugar .
*- Despite this small difference , A & B antigens do not cross – react
*- R.B.Cs have 3 terminal sugars ( in common ) on there are surface..
**-- Type A cells have an additional N –acetylgalatose .
**-- Type B cells have an additional galactose.
N.B . Type A & B genes code for transferaes that add
the respective Sugar .
*-* Type O have only the H antigen :
To avoid Ag – Ab reactions that would result in transfusion, all blood
for transfusion must be carefully cross matched .
*-* So , Ag the corresponding Ab do not coexist in the some person's
blood .
*-* Transfusion reactions result when incompatible donor's R.B.Cs
are transfused e . g . group A group B .
N – acetyl glucose amine
Galactose Fucose H antigen
In to
Ag – Ab – Reaction Involving R . B . Cs antigens
:-
ABO blood group . Structure of the terminal sugars that
Determine ABO blood groups .
4
POSSIBILITES
OF
CONBINATION
Antibody in plasma Antigen on R.B.Cs Group
Anti B A A
Anti A B B
No Anti ANOR Anti B A& B AB
Anti A & Anti B No A nor B O