2. We are exposed to a huge variety of
micro-organisms
The micro-organisms include bacteria,
viruses, parasites etc
Many of these are harmless; some are
even useful
3. There are many micro-organisms that
can cause disease
These are known as pathogenic micro-
organisms (or pathogens)
Diseases caused by them can be serious,
even fatal
4. Nature has equipped us with very
competent defense mechanisms
These mechanisms help us to deal with
the foreign invaders
6. This is a mechanism that we are born
with
Skin and mucous membranes act as
physical barriers
They prevent the entry of pathogens in
our body
Innate immunity
7. Skin and mucous membranes are
covered with some secretions
These secretions create an inhospitable
environment for pathogens
8. If a pathogen crosses the physical
barriers, it is challenged by phagocytes
Phagocytes include macrophages,
neutrophils etc
Phagocytes engulf and destroy the
micro-organisms
9. Besides phagocytes, some proteins also
help in fighting against pathogens
These include complement proteins,
acute phase proteins, interferons etc
10. The second component of the defence
mechanism is adaptive immunity
Unlike innate immunity, adaptive immunity
is acquired after birth
Adaptive immunity is also known as
acquired immunity
11. Innate immunity
Doesn’t recognize
antigens
Is non-specific
Doesn’t improve on
repeated exposure to
the same pathogen
Acquired immunity
Recognizes
antigens
Is specific
Improves on
repeated exposure to
the same pathogen
12. Acquired immunity is provided by some
cells and molecules
These cells and molecules recognize
chemicals called antigens
A given immune cell or molecule can
recognize only one antigen
Acquired immunity
15. Foreign chemicals having high molecular
weight
May be proteins, polysaccharides, nucleic
acids, synthetic chemicals
Have complex structure
May enter as free molecules or as
components of some cell
Antigens
16. Antigens can evoke an immune response
Immune response may be humoral or
cellular or both
A small part of the antigen is recognized
by the immune system
This part is known as epitope or antigenic
determinant
17. Low molecular weight compounds
Cannot evoke an immune response by
themselves
Can evoke an immune response when
combined with some other large molecule
Haptens
18. Once immune response develops, the
free hapten can be recognized by the
immune system
Some drugs act as haptens in some
individuals
20. Are the key cells of
immune system
Are formed from stem
cells in bone marrow
Differentiate into B
and T lymphocytes
Lymphocytes:
21. B lymphocytes (B cells) are processed in
bone marrow
B lymphocytes synthesize antibodies that
provide humoral immunity
T lymphocytes (T cells) are processed in
thymus
T lymphocytes provide cell-mediated
immunity
22. Both B lymphocytes and T lymphocytes
are capable of recognizing antigens
A given B cell or T cell can recognize only
one particular antigen
23. Operates against extracellular antigens
Operates through antibodies
Antibodies are plasma proteins belonging
to g-globulin fraction
They are also known as immunoglobulins
Humoral immunity
24. One light chain is joined to one heavy
chain by disulphide bonds
Heavy chains are joined to each other
by disulphide bonds
Two light chainsTwo heavy chains
Basic unit of an immunoglobulin (Ig)
molecule consists of:
25.
26. Immunoglobulin light chains
Light chains are smaller than heavy chains
Their molecular weight is about 23,000
They are of two types - k (kappa) and l
(lambda)
27. One Ig has only one type of light chains
In one type of light chain (k or l):
C-terminal half has a constant amino
acid sequence (constant region)
N-terminal half has a variable amino
acid sequence (variable region)
28. Heavy chains have a molecular weight of
50,000-70,000
They are of five types - a (alpha), d (delta),
e (epsilon), l (gamma) and m (mu)
Immunoglobulin heavy chains
29. One Ig has only one type of heavy chains
In one type of heavy chain:
C-terminal three-fourth has a
constant amino acid sequence
N-terminal one-fourth has a
variable amino acid sequence
30. C-Terminal three-fourth of heavy chain
is known as its constant region
N-Terminal one-fourth of heavy chain
is known as its variable region
31.
32. The light chains and heavy chains are
folded to form some globular domains
The variable regions of light and heavy
chains come together to form antigen-
binding sites
33.
34. Variable regions of light and heavy chains
have three hypervariable (HV) regions
Amino acid sequence is extremely variable
in hypervariable regions
HV regions constitute the antigen-binding
sites
35. HV regions are also called comple-
mentarity determining regions (CDRs)
They determine the complementarity
between the antigen and the antibody
36.
37. Only a small region of the antigen, known
as epitope, is recognized by the antibody
The region of antibody that contacts the
epitope is known as paratope
38. Action of papain
The proteolytic enzyme, papain cleaves
the antibody molecule
The molecule is broken up into:
Two identical Fab fragments
One Fc fragment
39. Each Fab fragment can bind antigen
(Fragment antigen binding)
Fc fragment can be easily crystallized
(Fragment crystallizable)
Papain treatment has thus shown the
functions of different regions of antibody
41. Antigen-antibody interaction
The antibody binds the comple-
mentary epitope of an antigen by:
Hydrogen bonds
Electrostatic bonds
Hydrophobic interactions
van der walls forces
42. Antigen-antibody binding is non-covalent
and reversible
After antigen binding, the constant (Fc)
region of the antibody performs the
effector functions
43. Effector mechanisms
Antibodies defend the organism by a
number of effector mechanisms
The important mechanisms are:
Neutralization
Opsonization
Complement activation
44. Antibodies coat the antigens present on
the surface of pathogens and prevent
their entry in cells
Antibodies also coat the toxins released
by pathogens and prevent their toxic
effects
Neutralization
48. Antibodies also attract natural killer (NK)
cells
NK cells cause antibody-dependent cell-
mediated cytotoxicity (ADCC) which kills
the pathogens
49. Binding of antibodies to antigens on the
surface of pathogens can activate the
complement system
Complement system either destroys the
pathogens directly or facilitates their
destruction by phagocytes
Complement activation
53. IgA is divided into two subclasses – IgA1
and IgA2
IgG has four subclasses – IgG1, IgG2,
IgG3 and IgG4
Subclasses
54. Most abundant Ig in plasma
Longest half-life
Performs all the effector functions
Can cross placental barrier
Maternal IgG provides immunity to the
newborn in the first few weeks of life
IgG
56. IgA is present in plasma as well as in
exocrine secretions
IgA present in exocrine secretions is
known as secretory IgA
Plasma IgA is a monomer while secretory
IgA is a dimer
IgA
58. Secretory IgA is formed beneath the
baso-lateral surface of epithelial cells
It binds to an Ig receptor present on
epithelial cells
A part of the Ig receptor detaches from
the cell
It attaches to IgA dimer as secretory
component
Secretory IgA
59.
60. Secretory IgA traverses the cell to reach
the mucosal surface
It acts mainly by neutralization
Secretory IgA present in breast milk
enters the gut of the newborn
It binds to mucosa and protects the baby
against gastro-intestinal pathogens
61. The largest Ig
The first Ig to be secreted upon entry of
any antigen
Acts mainly by complement activation
Weak neutralizing and opsonizing activity
IgM
62.
63. Bound to mast cells and basophils
Parasitic antigens bind to IgE on mast
cells
Mast cells release chemical mediators
that destroy the parasite
Also mediates allergic reactions
IgE
66. Important features of Ig classes
Class
of Ig
Plasma
conc (mg/dl)
Half-life
(days)
Active
against
Bacteria and
viruses
Strongly antiviral,
weakly antibacterial
Strongly antibacterial,
weakly antiviral
Anti-parasitic, mediates
allergic reactions
Function not
known
IgG
IgA
IgM
IgE
IgD
700-1,500
60-500
40-200
0.01-0.1
0.3-40
23
5-6
5-6
2-3
2-3
67. Variants of the basic
immunoglobulin structure are:
Idiotypes
Isotypes
Allotypes
Variants of immunoglobulins
68. The variable region of an Ig forms a
typical three-dimensional structure
This is complementary to the antigenic
determinant of a particular antigen
This typical antigen-recognition structure
constitutes an idiotype
Idiotypes
70. Immunoglobulins are divided into five
classes viz. IgA, IgD, IgE, IgG and IgM
IgA is further divided into IgA1 and IgA2
subclasses
IgG is further divided into IgG1, IgG2,
IgG3 and IgG4 subclasses
Isotypes
71. These variants (the classes and the
subclasses) are known as isotypes
Isotypic variations are due to differences
in amino acid sequence in the constant
region of heavy chains
73. Allotypes are immunoglobulins of the same
class differing slightly in amino acid sequence
in the constant region
These are formed by mutations substituting
one or two amino acids in the constant region
Allotypes are inherited from parents
Allotypes
75. Our immune system can produce a vast
range of antibodies having different
antigen specificities
These antibodies can recognize virtually
every actual and potential antigen that
we may come across
Antibody diversity
76. Every antibody has a unique antigen-
binding site
This is due to a unique amino acid
sequence in the variable region
Amino acid sequence of each protein is
encoded by a specific gene
77. Human beings can synthesize millions of
unique antibodies
So, we need millions of genes to encode
the huge repertoire of antibodies
However, the number of antibody genes
in human genome is less than 200
Every protein is encoded by a particular
gene
78. How a small number of genes
can produce a vast range of
antibodies remained a mystery
for long
Tonegawa solved this mystery
by showing that antibody
diversity arises from gene re-
arrangement
79. Genes for light chains and heavy chains
are present on different chromosomes
In genomic DNA, light chain and heavy
chain genes are not complete genes
The genes are split into segments
The segments are brought together
when a new B cell is being formed
Gene re-arrangement
80. Light chain gene re-arrangement
A light chain gene is made
up of three segments:
Variable (V) segment
Joining (J) segment
Constant (C) segment
81. Genes for V, J & C segments are present in
3 different clusters on two chromosomes
The genes for k light chains are present on
chromosome 2
The genes for l light chains are present on
chromosome 22
82. For k light chains, there are:
40 genes in V cluster
5 genes in J cluster
One gene in C cluster
83. For l light chains, there are:
30 genes in V cluster
4 genes in J cluster
4 genes in C cluster
84. The V segment and J segment genes
encode the variable region of the light
chains
The C segment genes encode the
constant region of the light chains
85. When a B cell differentiates, one of the V
segment genes joins one of the J
segment genes
The intervening DNA is deleted
This is known as V-J joining
86. The V-J combination and a C segment
gene are transcribed to form a hnRNA
hnRNA is spliced to form mRNA for a
light chain
88. There are 40 genes for V segment and 5
genes for J segment of k light chains
Therefore, 40x5 i.e. 200 different V-J
combinations are possible
89. There are 30 genes for V segment and 4
genes for J segment of l light chains
Hence, 30x4 i.e. 120 V-J combinations
are possible
A given B cell has one particular V-J
combination
90. Heavy chain gene re-arrangement
A heavy chain gene is made
up of four segments:
Variable (V) segment
Diversity (D) segment
Joining (J) segment
Constant (C) segment
91. The heavy chain genes are present in
four different clusters on chromosome 14
V, D and J segments encode the variable
region
C segment encodes the constant region
92. For heavy chains, there
are:
40 genes in V cluster
25 genes in D cluster
6 genes in J cluster
9 genes in C cluster
93. When a B cell differentiates, one D
segment gene joins one J segment gene
The intervening DNA is deleted
This is known as D-J joining
94. One V segment gene joins the D-J
combination
The intervening DNA is deleted
This is known as V-D-J joining
V-D-J combination and the first C segment
gene (Cm) are transcribed to form hnRNA
95. hnRNA is spliced to form heavy chain
mRNA
The V-D-J portion encodes the variable
region
Possible V-D-J combinations are
40x25x6 = 6,000
A given B cell has one particular
combination
97. Thus several hundred different light
chains can be formed
Each has a unique amino acid sequence
in its variable region
Several thousand different heavy chains
can be formed
Each has a unique amino acid sequence
in its variable region
98. When light chains combine with heavy
chains, millions of combinations are
possible
Each combination is specific for one
antigen
A given B cell has only one combination;
it synthesizes only one specific antibody
99. Variable combination of different gene
segments is the major mechanism of
antibody diversity
Antibody diversity, thus, generated is
known as combinatorial diversity
Nearly two million variable regions can be
formed by combinatorial diversity
Combinatorial diversity
100. Gene re-arrangement reactions are
catalyzed by:
DNA repair enzymes
RAG-1 and RAG-2
RAG-1 and RAG-2 are encoded by
recombination activating genes 1 and 2
101. A second source of diversity is junctional
diversity
When different gene segments join, some
nucleotides may be added to, or deleted
from, the ends
This creates new base sequences
Junctional diversity
102. Nucleotides are added by Terminal
deoxyribonucleotidyl Transferase (TdT)
Nucleotides are deleted by exonucleases
103. A final source of diversity is somatic
hypermutation
When a mature B cell encounters an
antigen, a series of point mutations can
occur in the variable region of the gene
This results in further diversity
Somatic hypermutation
104. Like other genes, the heavy chain genes
are preceded by a promoter
A regulator is present upstream of the
promoter
An enhancer element is present between
V-D-J and C segments
Re-arranged heavy chain gene
105. There are nine C (constant) segments
The promoter is weak but gene expression
is increased by the enhancer element
Each C segment is preceded by a short
repetitive sequence known as switch
sequence
106.
107. Among the nine C segments, the first is Cm
IgM is the first immunoglobulin to be
formed upon entry of any antigen
After V-D-J joining, VDJ is transcribed with
the first constant segment i.e. Cm
108. Different classes of immunoglobulins differ
in their heavy chains and effector functions
IgM is the first immunoglobulin to be
formed
However, Ig of a different class may be
needed to deal with the same antigen
Class switching
109. The new Ig should recognize the same
antigen but perform a different effector
function
For this, the VDJ complex, already formed,
joins a downstream constant segment
The intervening DNA is deleted
110. Thus, an Ig of a different class but having
the same antigen specificity is formed
This is known as class switching (isotype
switching)
Switch sequences preceding C segment
genes facilitate class switching
112. There are millions of B cells, each
synthesizing one particular Ig
The Ig molecules are inserted in the cell
membrane of B cells
These are known as antigen receptors
The antigen-binding site is displayed on
the cell surface
Primary response
113.
114. The antigens possess specific epitopes
Each epitope is recognized by a
complementary antigen receptor
115.
116. When an antigen enters for the first time, its
epitope is recognized by some Ig molecule
The Ig molecule binds the antigen
This binding stimulates the B lymphocyte to
divide and differentiate
The B cell differentiates into a clone of
plasma cells
117.
118. The plasma cells not only synthesize but
also secrete the antibody
Antibody secretion upon first exposure to
an antigen is called primary response
Primary response is weak and short-lived
119. After first encounter with an antigen, some
B cells are converted into memory B cells
If the same antigen enters again, memory
B cells differentiate into plasma cells
This is called secondary response
Secondary response is quick, strong and
long-lasting
Secondary response
120.
121. Immunoglobulins synthesized by B cells
are inserted in their cell membrane
B cells cannot secrete immunoglobulins
into plasma
Immunoglobulins displayed on the surface
of B cells are known as antigen receptors
Differentiation of B cells into
plasma cells
122.
123. The antigen receptor inserted in the
membrane of B cells is IgM
The CH4 domain of membrane-bound IgM
is associated with two trans-membrane
proteins
These are Ig-a and Ig-b
124. Cytoplasmic portions of Ig-a and Ig-b
possess immunoreceptor tyrosine-based
activation motif (ITAM)
ITAM is made up of two precisely spaced
tyrosine residues that are 9-11 amino acid
residues apart
125. When an antigen enters the body, its
epitope finds a complementary antigen
receptor
The antigen binds to the receptor
Antigen-binding causes oligomerization of
neighbouring IgM molecules
126. Oligomerization activates an intracellular
tyrosine kinase, Fyn
Some other cytosolic tyrosine kinases
(Lyn and Blk) may also be activated
The activated Fyn phosphorylates the
ITAMs of Ig-a and Ig-b
127. The phosphorylated ITAMs act as a
docking site for a protein, Syk
Syk, which is also a tyrosine kinase, is
activated
Active Syk phosphorylates the tyrosine
residues of some other target proteins
128.
129. The phosphorylated target proteins
generate some signals
These signals stimulate the B cell to
differentiate into a clone of plasma cells
Plasma cells synthesize IgM of a different
kind
130. IgM can be of two types -
membrane-bound form and secreted form
The two forms are synthesized from the
same hnRNA by alternative splicing
The splicing which occurs in B cells
produces the membrane-bound form
The splicing which occurs in plasma cells
produces the secreted form
131. Immunization is a process by which active
immunity against an infection is produced
This is done by administering a vaccine
Vaccine contains a small dose of an
antigen belonging to the infectious agent
Immunization
132. The antigen in the vaccine produces a
primary response
The immune system retains the memory
of the antigen
133. If the same antigen enters again by way
of natural infection, a quick secondary
response develops
The secondary response destroys the
antigen (and the infectious agent)
Immunization is used to prevent a variety
of infectious diseases
134. The vaccine may consist of:
• Killed micro-organisms e.g. typhoid
vaccine, cholera vaccine, Salk’s polio
vaccine etc
• Live attenuated micro-organisms e.g.
Sabine’s polio vaccine, BCG vaccine etc
• Toxoids e.g. tetanus toxoid, diphtheria
toxoid etc
• Pure antigen e.g. Hepatitis B surface
antigen in recombinant hepatitis B vaccine
135. Features of an effective vaccine
Safe
The vaccine itself should
not cause disease or
death
Protective
Must protect against
illness from exposure to
live pathogen
Sustained
effect
Protection should be
long-lasting
136. Passive immunity
Quick but short-term protection against
an infection can be provided by
administering pre-formed antibodies
These antibodies may be:
Obtained from animals
Obtained from human beings
Synthesized in the laboratory
137. Monoclonal antibodies produced in
laboratory by hybridoma technology
are widely used for:
Academic purposes
Diagnostic purposes
Therapeutic purposes
138. Monoclonal antibodies are clones of each
other
They are produced in the laboratory by
hybridoma technology
A hybridoma cell is a hybrid cell prepared
by fusion of a B lymphocyte and a
myeloma cell
Production of monoclonal antibodies
139. B lymphocytes are antibody-forming cells
A given B lymphocyte is dedicated to form
one particular antibody
Myeloma cells are immortal cancer cells
Fusion of a B lymphocyte with a myeloma
cell produces a hybridoma cell
140. Hybridoma cell possesses properties of
myeloma cells as well as B cells
A hybridoma cell is immortal like a
myeloma cell
It synthesizes one particular antibody like
a B cell
141. When the hybridoma cell is put in a
culture medium, it goes on:
Dividing for ever
Secreting the antibody for ever
Since only one B-lymphocyte has fused
with a myeloma cell, the hybridoma cell
secretes a monoclonal antibody
142. A mouse is inoculated with an antigen
B cells are taken from its spleen
B cells and myeloma cells are fused in the
presence of polyethylene glycol (PEG)
Procedure
143. When B cells and myeloma cells are
put together in the presence of PEG:
• Some B cells may fuse with B cells
• Some myeloma cells may fuse with
myeloma cells
• Some B cells may fuse with myeloma
cells
• Some B cells and myeloma cells may
remain unfused
144. Fusion of B cells with myeloma cells
produces hybridoma cells
Hybridoma cells have to be selected from
the other cells
This is done by culturing the cells in
Hypoxanthine-Aminopterin-Thymidine
(HAT) medium
146. Myeloma cells lack HGPRT
Therefore, they cannot salvage hypo-
xanthine (present in HAT medium)
They are dependent on de novo synthesis
for their purine requirement
But de novo synthesis is blocked by
aminopterin
147. Unfused B-lymphocytes die soon when
their life-span is over
Unfused myeloma cells cannot survive
because they are deprived of purines
Only hybridoma cells survive as they have
acquired HGPRT from B-lymphocytes
In HAT medium:
148. Myeloma cells cannot
salvage hypoxanthine
as they lack HGPRT
De novo synthesis of
purine nucleotides and
TMP is blocked by
aminopterin
PRPP
AMP←IMP→GMP
HGPRT
Hypoxanthine
↓
↓
↓
↓
↓
UMP
TMP
↓
↓
↓
↓
↓
Thymidine
Thymidine
kinase
Production of monoclonal antibodies
‒‒
149. Complement system acts in concert with
antibodies
It complements the action of antibodies
It consists of a group of proteins present
in plasma
Complement system
153. The small peptide split off is denoted by
suffix ‘a’ and the larger fragment by ‘b’
The enzymatically active fragment is
shown by a horizontal bar on it
X → Xā + Xb
154. There are three complement pathways:
Classic complement cascade
Lectin pathway
Alternate complement pathway
155. The classic complement cascade is
activated by antigen-antibody binding
Lectin and alternate complement pathways
don’t require antigen-antibody interaction
156. Classic complement cascade
The classic complement cascade
can be divided into three phases:
Recognition phase
Activation phase
Membrane attack phase
159. Membrane
attack phase
A membrane attack complex
participates in this phase
The membrane attack complex is
made up of C5, C6, C7, C8 and C9
160. Binding of IgM or IgG to a bacterial
antigen initiates the recognition phase
One IgM or at least two (ideally six) IgG
molecules are required for initiation
Binding of IgM or IgG to the antigen
attracts C1
Recognition phase
162. C1 is a complex made up of C1q, C1r and
C1s
C1q consists of six identical subunits with
globular heads and long tails
The tails combine to bind two molecules
each of C1r and C1s
163.
164. C1 binds to the constant region of the
antibody
This binding causes a conformational
change in C1r
As a result, C1r cleaves and activates
C1s
C1s → C1s
165.
166. C6 + C7
C3b
C5b
C5b-6-7
C5 C5a
C8
C5b-6-7-8
Membrane attack phase
C5b, C6, C7 and C8 combine to form
the membrane attack unit
C3b splits C5 into C5a and C5b
The unit inserts in the cell membrane of
the target cell and attracts C9
167. Several molecules of C9 insert in the cell
membrane of the target cell
They are polymerized to form a trans-
membrane annular pore
Contents of the cell leak out through the
pore leading to lysis of the target cell
168.
169. The complement pathway can also be
activated by mannose binding lectin (MBL)
MBL is a protein synthesized by liver and
secreted in circulation
MBL binds to mannose (and some other
carbohydrates) of microbes
Lectin pathway
170. Binding of MBL to microbial
carbohydrates activates MBL-associated
serine protease (MASP)
MASP activates complements C2 and C4
Rest of the pathway is identical with the
classic complement pathway
173. The alternate complement pathway does
not involve antigen-antibody interaction
It does not require C1, C2 and C4
It requires the rest of the complement
components and Factor B and Factor D
Alternate complement pathway
174. According to current view, C3 is slowly
hydrolysed when it contacts water
This slow and spontaneous hydrolysis
occurs continuously
If a foreign cell is not present, active C3b
binds to some self-cell
175. C3 is inactivated by factors H and I present
on the self-cell
If a foreign cell is present, C3 binds to the
foreign cell
C3 remains active because the foreign cells
lack factors H and I
176. A protein, Factor B binds to C3b to
form a complex
Factor D splits Factor B into Ba and Bb
C3bBb splits C5 into C5a and C5b
180. Allergy is described as a side effect of
immunity
It is an altered immune response to an
otherwise innocuous antigen
Antigens evoking an allergic response
are called allergens
Allergy
181. Allergens are small molecules
present in:
• Pollens
• Molds (tiny fungi)
• Dust mites
• Animal dander (flecks of skin)
• Insect sting
• Drugs
• Food
182. Allergens bind to IgE (Reaginic antibody)
IgE is normally present in minute
concentration
Its function is to protect against parasites
183. Some persons have a relatively high
concentration of IgE
The concentration is influenced by genetic
as well as environmental factors
184. Genetic susceptibility, inherited from
parents, can increase IgE concentration
Very hygienic environment spares IgE as
it doesn’t have to act against parasites
185. High-affinity receptors for IgE are present
on mast cells
Mast cells are present beneath mucosal
cells and in connective tissue
IgE binds to its receptors on the mast cells
186. When an allergen binds to IgE, the mast
cells release their stored granules
The granules contain some chemical
mediators
187. The chemical mediators include:
Histamine
Heparin
Leukotrienes C4, D4 and E4
Platelet activating factor
Eosinophil chemotactic factor
188. Increase in capillary permeability
Vasodilatation
Itching
Sneezing
Bronchospasm
Depending upon the site of release,
chemical mediators cause: