MB 504 Basic Immunology

KBCNMU conducted Science & Commerce College, Molgi
B.Sc. T.Y. MICROBIOLOGY

MB 504-BASIC IMMUNOLOGY
UNIT 1 HISTORY OF IMMUNOLOGY

The characteristics of antigens
Antigens are large molecules of proteins, present on the surface of the pathogen, like bacteria,
fungi viruses, and other foreign particles. When these harmful agents enter the body, it induces
an immune reaction within the body for the assembly of antibodies.
For Example: When a standard cold virus enters the body, it causes the body to supply
antibodies to stop from getting sick.
Properties of Antigens
The properties of antigens are as follows:
 The antigen should be a far off substance to induce an immune reaction .
 The antigens have a molecular mass of 14,000 – 6,00,000 Da.
 They are mainly proteins and polysaccharides.
 The more chemically complex they are, the more immunogenic they will be.
 Antigens are species-specific.
 Age influences immunogenicity. Very young and really old people exhibit very low
immunogenicity.
Types of Antigens
On the Basis of Origin
There are different types of antigens on the idea of origin:
Exogenous Antigens
These are the antigens that have entered the body from outside, for example, inhalation,
injection, etc. These are the foremost common sorts of antigens and include pollens and food
that cause allergies.
Endogenous Antigens
DR SHAKYASHING JADHAV ASSISTANT PROF IN

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Endogenous antigens are people who are generated within the traditional cells thanks to viral or
intracellular bacterial infections.
Autoantigens
Autoantigens are normal proteins or a posh of proteins or macromolecules that's attacked by a

system causing autoimmune diseases.
Tumour Antigens
It is an antigenic substance produced in tumour cells that induces an immune reaction within
the host. These are presented by MHC-I and MHC-II on the surface of tumour cells.
Native Antigens
A native antigen is the one that's not processed by an antigen-presenting cell.
On the Basis of Immune Response
On the idea of the immune reaction , antigens are often classified as:
Immunogen
These could also be proteins or polysaccharides and may generate an immune reaction on their
own.
Hapten
These are non-protein, foreign substances that need a carrier molecule to induce an immune
reaction.
Antibodies aren't found at an area intrinsically, but whenever our system encounters antigen of
a pathogen, B cells get activated immediately releasing antibodies into the bloodstream. These
immunoglobulins undergo mitosis leading to cellular division and continuously produce
antibodies as a result of producing more cells. These antibodies remain within the blood for a
few times but B cells remember these antigens and repeat an equivalent course of action
whenever they reappear in our body.
EPITOPES
The small site on an antigen to which a complementary antibody may specifically bind is called
an epitope or antigenic determinant. This is usually one to six monosaccharides or five to eight
amino acid residues on the surface of the antigen. Because antigen molecules exist in space, the

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epitope recognized by an antibody may be dependent upon the presence of a specific three
dimensional antigenic conformation (e.g., a unique site formed by the interaction of two native
protein loops or subunits). This is known as a conformational epitope. The epitope may also
correspond to a simple linear sequence of amino acids and such epitopes are known as linear
epitopes.
The range of possible binding sites on a target molecule (antigen) is enormous, with each
potential binding site having its own structural properties derived from covalent bonds, ionic
bonds, hydrophilic, and hydrophobic interactions. Indeed, this has important ramifications for
antibody choice and performance. For efficient interaction to occur between the target antigen
and the antibody, the epitope must be readily available for binding.
If the target molecule is denatured, e.g., through fixation, reduction, pH changes, or during
preparation for gel electrophoresis, the epitope may be altered and this may affect its ability to
interact with an antibody. For example, some antibodies are ineffective in Western blotting
(WB) but are suitable for immunohistochemistry (IHC) applications, because, in the IHC
procedure, a complex antigenic site might be maintained in the tissue, whereas in the WB
procedure, the process of sample preparation alters the protein conformation sufficiently to
destroy the antigenic site, and hence eliminates antibody binding.
In a denatured protein, only the linear epitope may be recognized. Hence, in protocols where a
denatured protein is used, such as in Western blotting, an antibody that recognizes a linear
epitope is preferred. Sometimes an epitope is on the interior of a folded protein. The epitope is
then inaccessible to the antibody in a nondenaturing protocol, such as immunoprecipitation. A
conformational epitope, by definition, is on the outside of the folded protein. An antibody that
recognizes the conformational epitope is suitable for mild, nondenaturing procedures, such as
immunoprecipitation or flow cytometry.
Optimally, an antibody that recognizes a linear epitope on the surface of a normally folded
protein will work well in both nondenaturing and denaturing protocols. Thus, the epitope may
be present in the antigen’s native, cellular environment, or it may be exposed only when
denatured. In their natural form, antigens may be cytoplasmic (soluble), membrane-associated,
or secreted. The number, location and size of the epitopes depend on how much of the antigen
is presented during the antibody-making process.
Knowledge about the target protein, the epitope recognized by the antibody, sequence
conservation, and the technique principles are valuable in making good antibody and protocol
choices. Actual epitope mapping or sequence data, though useful, are not needed, however, to
be confident in antibody specificity.

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Difference Between Antigen and Antibody
Antigen Antibody
Also called Immunogens Also called Immunoglobulins
Antigens are usually lipids. However, they

can also be proteins, carbohydrates, and All antibodies are proteins
nucleic acids
Protects against the effects of the

Antigens cause allergic reactions or even
antigen either by lysis or immobilization
illnesses
of the particle
Epitopes are regions of the antigen where Paratopes are variable regions of an
interacts with the antibodies antibody that binds to an epitope.
Antibodies always originate within the

Antigens have origins outside the body
body
What are Antibodies?
Antibody (Ab) is also known as an immunoglobulin(Ig). These are big in size, Y-shaped blood
proteins produced by plasma cells. They bind to foreign particles and invade them. Antigens are
foreign pathogens that invade the body and have the potential to offer rise to a response from
our immunity system either by grouping up with a bigger molecule or alone after binding with
antibodies for a specific immune reaction. Hence, antigens stimulate the assembly of antibodies
by the system.

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Antibody Structure
An antibody structure consists of a Y-shaped molecule composed of 4 polypeptide subunits. The

N-terminus of the heavy chains associates with one of the light chains and forms two antigen
binding domains. These form the arms of the “Y” shape and are referred to as fragment antigen
binding (Fab) domains. It helps within the antibody’s interaction with the effector cells.
All four polypeptide subunits are held together by disulfide and non-covalent bonds.
The large chains of the antibodies contain a variable region and three constant regions. Each
antibody has two identical antigen-binding sites and they differ within the antibodies.
What is an Antibody (Ab)?
An antibody is simply the component produced by the immune system in response to antigens.
So basically antigens are the generator of antibodies. They interact with each other to induce an
immune response.
– Also called immunoglobulins (Ig)
– Y-shaped
– Glycoproteins
– Produced by plasma B-cells
– Antigen binding site is called paratope.
– Types: IgG, IgA, IgM, IgE, IgD (Pneumonics: “GAMED”)
What is an Antigen-Antibody Interaction?
The interactions between antigens and antibodies are known as antigen-antibody reactions.
The reactions are highly specific, and an antigen reacts only with antibodies produced by itself
or with closely related antigens. Antibodies recognize molecular shapes (epitopes) on antigens.
Generally, the better the fit of the epitope (in terms of geometry and chemical character) to the
antibody combining site, the more favorable the interactions that will be formed between the
antibody and antigen and the higher the affinity of the antibody for antigen. The affinity of the
antibody for the antigen is one of the most important factors in determining antibody efficacy in
vivo.

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The antigen-antibody interaction is a bimolecular irreversible association between antigen and

antibody. The association between antigen and antibody includes various non-covalent
interactions between epitope (antigenic determinant) and variable region (VH/VL) domain of
an antibody.
Antigen-Antibody Interaction Properties
– Highly specific reaction
– Occurs in an observable manner
– Non-covalent interaction ( Van der Waals forces, Ionic bonds, Hydrogen bonds, Hydrophobic
interactions )
– No denaturation of antibodies and antigens
– Reversible
– Affinity: It is the strength with which one antigen binds on a single antigen-binding site on an
antibody.
– Avidity: It is a broader term than affinity. It is a measure of the overall strength of the Ag-Ab
complex. It depends on:
 The affinity of the antibody
 Valency(no. of binding sites) of antibody and antigen
 And the structural arrangement of epitopes and paratopes.
– Cross-Reactivity: It refers to the ability of an antibody to bind to similar epitopes of different

antigens.
Stages of Antigen-Antibody Interaction
1. Primary Stage: It is the initial interaction between antigens and antibodies.
– Rapid
– Reversible
– Without any visible effects
2. Secondary Stage: It is the irreversible interaction between antigens and antibodies.

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– Slow
– With visible effects
Factors Affecting Antigen-Antibody Interaction
Many factors affect Ag-Ab reactions. Some of the common factors are:
1. Temperature: It depends on the chemical nature of epitopes, paratopes, and, bonds involved.
Eg. hydrogen bonds are stable at low temperatures and hydrophobic bonds are stable at high
temperatures.
2. pH: Optimal pH range is 6.5 to 8.5. Extreme pH values change the conformation of antibodies
and inhibit the reaction.
3. Ionic strength: The effect of ionic is important in blood group serology. Here the reaction is
significantly influenced by sodium and chloride ions. In normal saline solution, Na+ and Cl−
cluster around the complex and partially neutralize charges, potentially interfering with
antibody binding to antigen. This could be problematic when low-affinity antibodies are used.
4. Enzyme Treatment: Many proteolytic enzymes are used to enhance the Ag-Ab reactions. Some
of the commonly used ones are papain, ficin, bromelin.
5. Concentrations of Ag and Ab: Increase in the concentration of antigen and antibody enhances

the reaction.
6. No. of antigen-binding sites: More the no. of antigen-binding sites on the antibody, the more
the chances of interaction. For eg., IgM is a pentamer and hence has 10 binding sites whereas
IgG is a monomer and hence has only 2 binding sites so IgM will bind more efficiently with
antigens.
7. Structural arrangement: If the structure of epitope and paratope is such that they could fit well
as lock and key then it enhances the interaction between antigen and antibody.
Chemical Bonds Responsible for the Antigen-Antibody Reaction
The interaction between the Ab-binding site and the epitope involves exclusively non-covalent
bonds, in a similar manner to that in which proteins bind to their cellular receptors, or enzymes
bind to their substrates. The binding is reversible and can be prevented or dissociated by high
ionic strength or extreme pH. The following intermolecular forces are involved in Ag–Ab binding:
1. Electrostatic bonds: This results from the attraction between oppositely charged ionic groups of
two protein side chains; for example, an ionized amino group (NH4+) on lysine in the Ab, and an
ionized carboxyl group (COO_) on an aspartate residue in the Ag.

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2. Hydrogen bonding: When the Ag and Ab are in very close proximity, relatively weak hydrogen
bonds can be formed between hydrophilic groups (e.g., OH and C=O, NH, and C=O, and NH and
OH groups).
3. Hydrophobic interactions: Hydrophobic groups, such as the side chains of valine, leucine, and
phenylalanine, tend to associate due to Van der Waals bonding and coalesce in an aqueous
environment, excluding water molecules from their surroundings. As a consequence, the
distance between them decreases, enhancing the energies of attraction involved. This type of
interaction is estimated to contribute up to 50% of the total strength of the Ag–Ab bond.
4. Van der Waals bonds: These forces depend upon interactions between the “electron clouds”
that surround the Ag and Ab molecules. The interaction has been compared to that which might
exist between alternating dipoles in two molecules, alternating in such a way that, at any given
moment, oppositely oriented dipoles will be present in closely apposed areas of the Ag and Ab
molecules.
Each of these non-covalent interactions operates over a very short distance (generally about 1
Å) so, Ag-Ab interactions depend on a very close fit between antigen and antibody.
Strength of Ag-Ab interactions
1. Affinity
 The combined strength of total non-covalent interactions between a single Ag-binding site of Ab

and a single epitope is the affinity of Ab for that epitope.
 Low-affinity Ab: Bind Ag weakly and dissociates readily.
 High-affinity Ab: Bind Ag tightly and remain bound longer.
2. Avidity
 The strength of multiple interactions between multivalent Ab and Ag is avidity. Avidity is a better
measure of the binding capacity of antibodies than affinity. High avidity can compensate for low
affinity.
3. Cross-reactivity
 Antibodies elicited by one Ag can cross-react with unrelated Ag if they share identical epitopes

or have similar chemical properties.
Types of Antigen-Antibody Interaction
Ag-Ab reactions are basically of two types:
1. In Vivo (Occurring in natural condition): It includes the general antibody-mediated immune

response occurring in our body against any antigen.

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2. In Vitro (Done in artificial conditions): It includes a series of serological tests performed in
laboratories to detect antigens or antibodies in case of many diseases.
In Vivo Reactions
 Agglutination
 Precipitation
 Complement fixation
 Neutralization
 Ab Dependent Cell-Mediated Toxicity
 Immobilization
 Opsonization
In vitro Reactions
 Agglutination
 Precipitation
 Complement fixation
 Neutralization
 ELISA
 Radioimmunoassay (RIA)
 Western Blotting
 Immunochromatograhy (ICT)
 Immunofluorescence
Precipitation Reaction
The reaction between soluble (small) antigens and soluble antibodies forms an insoluble
precipitate. The proportion of Ag and Ab in the reaction must be equivalent for the reaction to
occur.
– In the presence of electrolytes
– At specific pH and Temperature
– Antibodies involved are called precipitins
– Types: In solution (Flocculation and Ring Test)

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In Agar (Immunodiffusion)
In Agar with an electric field (Immunoelectrophoresis)
Agglutination Reaction
The reaction between insoluble (large) antigens and soluble antibodies leads to agglutination.
– Formation of visible clumps occurs.
– Occurs on the surface of the particle involved.
– Antibodies involved are called agglutinins.
– Types: Direct/Active (Slide, Tube, Heterophile agglutination test and, Coombs’ test,)
: Passive (Latex, Haemagglutination and, Coagglutination test)
Haemagglutination
It is a passive agglutination reaction that involves RBCs as carrier particles.
– RBCs of sheep, humans, chicks, etc. are used
– Used in Blood Typing
– In the detection of parasitic infections and viral diseases such as influenza, mumps, and
measles.
Note: In the case of sensitivity agglutination reaction is more sensitive than precipitation
reaction.
Complement Fixation
It is the process of binding or fixing the serum complement proteins with the Ag-Ab complex
which further initiates a series of immune responses against the antigen. It is used in the
detection of any specific antigen or antibody in the patient’s serum.
– Reagents: Antigen, Antibody, Complement, and, Indicator System
– Common complement fixation test is Wasserman’s Test: For detection of Syphilis
Enzyme-Linked Immunosorbent Assay (ELISA)
It is one of the sensitive techniques for the detection of the presence of antigen or antibody and
quantification as well in case of clinical diagnosis of many diseases such as AIDS, Ebola,
Pernicious anemia, different parasitic infections, etc. Enzymes are used for labeling.
– Detects free antigens or antibodies.

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– Reagents: Coating buffer, Blocking buffer, Wash Buffer, Substrates, Sample and, Assay
Diluents.
– Types: Direct, Indirect, and, Sandwich ELISA.
Hemolysis
When Ag-Ab interactions result in the rupture or lysis of RBC, it is called hemolysis which results
in the release of Haemoglobin. It can be catalyzed by enzymes called hemolysins. It is the
demonstrable endpoint of some reactions.
Immunofluorescence
It is a type of immunoassay technique in which fluorescent dyes are used for the visualization of
Ag-Ab reactions.
– Detects surface antigens or antibodies.
– Fluorescent dyes such are fluorescein isothiocyanate and lissamine rhodamine used.
– Types: Direct and Indirect.
Neutralization
In neutralization, the biological effects of viruses and toxins are neutralized by homologous
antibodies called neutralizing antibodies.
Types:- Virus Neutralization Tests (Eg. Viral hemagglutination inhibition test)
– Toxin Neutralization Tests (Eg. Schick test, antistreptolysin O test, etc.)
Radioimmunoassay (RIA)
It is a type of immunoassay in which radioisotopes are used for labeling the antigen or antibody
to detect the formation of the Ag-Ab complex.
– Can determine very small quantities of Ag and Ab in the serum.
– Used for the quantification of hormones, drugs, and, viral antigens.
Sensitization
The first step in the Ag-Ab interactions involves the formation of the Ag-Ab complex and it is
called sensitization. It is not observable and is observed only after agglutination of the formed
complex using different reagents. IgG antibodies can sensitize red cells to the corresponding
antigens and hence are called sensitizing antibodies.
Western Blotting
It is called so because of its similarity to Southern Blotting.

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– Protein separation is done by electrophoresis.
– Used in the detection of proteins.
– Confirmatory test in the diagnosis of HIV.
Antibody Definition
Antibodies, also known as immunoglobulins, are proteins produced by lymphocytes as a result

of interaction with antigens. Antibodies are a part of the humoral immune of the adaptive
immune system where each antibody identifies a specific antigen and protects the body
against it.
 Antibodies are glycoproteins that bind to antigens with a high degree of specificity and affinity.
 B lymphocytes are stimulated by the binding of antigen, which results in the secretion of

millions of antibodies in the bloodstream.
 The produced antibodies circulate through the bloodstream and neutralize antigens that are
identical to those that triggered the immune response.
 The binding of antibodies to microorganisms or other such antigens can result in the
microorganism being immobile or preventing them from penetrating the cells.
 Antibodies carry out two principal functions in the immune system. The first function is the
recognition and binding to foreign bodies. The second more important function is to trigger the
elimination of the attached foreign material.
 Since millions of antibodies are produced during an immune response, some of these remain in
circulation in the blood for several months. This provides an extended immunity against the
particular antigen.
 Each antibody is a Y-shaped protein where each tip of the Y contains a paratope that recognizes
an epitope of a particular antigen.
 Antibodies can be classified into different classes based on different structures and functions.

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Antibody Structure
Figure: Structure of an antibody
 Antibodies are globular plasma proteins with a basic Y-shaped structure and four polypeptides.
 There are two identical heavy chains and two identical light chains connected together by
disulfide bonds. The light chains consist of polypeptides of the size 22,000 Da, and the heavy
chains consist of polypeptides of the size 50,000 Da.
 Each heavy chain is connected to a light chain by a disulfide bond, and the two heavy chains are
connected to the light chains by two sulfide bonds.
 There are five different types of heavy chains in mammals that are designated by letters: α, δ, γ,
ε and µ. There are two types of light chains designated by λ and κ.
 An antibody is composed of a variable region and a constant region. The variable region changes
to various structures depending on the differences in the antigens. The constant regions are
constant and do not change with antigens.
 The variable region varies between clones and is involved in antigen recognition. The constant
region is conserved among clones and is required for the structural integrity and effector
functions.
 The heavy and light chain in an immunoglobulin molecule consists of an amino-terminal variable
region with 100-110 amino acids.
 Each heavy chain has one variable domain and one constant domain. The light chain, in turn,
consists of one variable domain and three constant domains.

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 The rest of the chain consists of the constant region, which limited variation that determines the
light chain subtypes.
 The heavy chains in some antibodies contain a proline-rich hinge region. The hinge region
separates the antigen-binding and effector domains.
 The region allows the movement of the domains enabling them to bind to antigens separated by
varying distances.
Antibody Types or Classes
 Antibodies can be classified into five different classes; IgG, IgM, IgA, IgD, and IgE. All the
antibodies have the basic four-chain antibody structure, but they have different heavy chains.
 The differences in the immunoglobulins are more pronounced in the Fc regions of the antibody,
which leads to the triggering of different effector functions.
 The structural differences in the antibodies also result in differences in the polymerization state
of the monomer unit.
 The following are the five different classes of antibodies;
Immunoglobulin G (IgG)
IgG is the most abundant immunoglobin, which accounts for about 80% of the total serum
antibodies. The concentration of IgG in the blood is about 10mg/ml.
Structure of IgG
 The basic structure of IgG is composed of a Y-shaped protein where the Fab arms are linked to
the Fc arms by an extended region of polypeptide chain called the hinge.
 The region is exposed and sensitive to attack by proteases that cleave the molecule into distinct
functional units arranged in a four-chain structure.

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 An IgG molecule consists of two identical γ heavy chains, usually of the size 50kDa.
 The light chains in IgG exist in two forms; κ and λ, where the κ form is more prevalent than λ, in
the case of humans.
 The Fc regions of the molecule have a highly conserved N-glycosylation site in the heavy chain.
Properties of IgG
 The IgG antibodies exist in the serum in the

monomeric form, and these can cross the placenta
from the mother to the fetus.
 Each IgG antibody has two paratopes that bind to two

different epitopes on different antigens.
 IgG has four subclasses classified on the basis of the

subclasses of the γ heavy chains.
 IgG antibodies participate predominantly in secondary

immune response as these are generated as a result of
class switching and maturation of the response.
Subclasses of IgG
 IgG antibodies have been classified into four subclasses; IgG1, IgG2, IgG3, and IgG4.
 These are named in the order of their abundance in serum, with IgG1 being the most abundant.
IgG1
 IgG1 is the most abundant subclass of IgG antibodies with γ1 heavy chains.
 IgG1 is primarily induced by soluble protein antigen and membrane proteins but is often
accompanied by lower levels of the other subclasses.
 A deficiency of IgG1 can lead to a decreased total IgG level as it is the most abundant subclass.
IgG2
 IgG2 is the second most abundant IgG in the human serum, and it is composed of γ2 heavy
chains.
 IgG2 is almost entirely responsible for the response against bacterial capsular polysaccharide
antigens.
 IgG2 is the only subclass of IgG antibodies that cannot cross the placenta during pregnancy.
 The deficiency of IgG2 can result in a weak defense against pathogenic microorganisms.

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IgG3
 IgG3 is the third most abundant IgG occurring in human serum with the γ3 heavy chains.
 These are particularly effective in the induction of effector functions. It is a potent

proinflammatory antibody with a shorter half-life.
 IgG3 is also the most effective complement activator and has a high affinity for FcR on
phagocytic cells, aiding in opsonization.
IgG4
 IgG4 is the least abundant IgG antibody in human serum, which consists of γ4 subclasses of
heavy chains.
 IgG4 is induced by allergens, and it is formed after repeated or long-term exposure to antigen in
a non-infectious setting.
 IgG4 can cross the placenta and transfer from the mother to the fetus. IgG4 deficiencies are
quite rare, but the increased levels of IgG4 in the serum have been associated with a number of
problems in different organs.
Functions of IgG
 IgG antibodies provide long-term protection against various agents like bacteria, viruses, and
bacterial toxins.
 IgG is one of the most potent complement activators when compared to all other antibodies.
 The binding ability of IgG to antigens is more effective as it enhances phagocytosis.
Immunoglobulin M (IgM)
IgM is the third most abundant immunoglobulin in serum, with a concentration of 1.5 mg/ml. It
is the largest antibody and is the first antibody to appear in response to the initial exposure to
antigen.
Structure of IgM
 IgM is secreted in a pentameric form with five distinct units, where each are composed of two µ
heavy chains and two light chains.
 A J chain might be present in the hexameric form of the molecule, but it isn’t always present.
The J chain is usually added just before the secretion of the pentamer as it helps in the
polymerization of the monomers.
 Each of the monomers has two antigen-binding sites, resulting in 10 binding domains in a single
molecule. However, all the domains cannot be occupied at the same time due to limitations in
space.

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 The pentameric form of IgM has a molecular weight of 900 kDa.
Properties of IgM
 IgM is the largest and the only pentameric antibody

in humans. It is also the first antibody to be produced
in response to the initial exposure to an antigen.
 IgM is the first immunoglobulin to be synthesized by

the fetus, beginning at about 20 weeks of age.
 IgM is a pentameric molecule with 10 antigen-

binding sites and 5 Fc portions held together by
disulfide linkages.
 The monomeric form of IgM occurs as the major

antibody receptor on the surface of B lymphocytes.
 IgM is relatively short-lived and usually disappears

earlier than IgG.
 The large size of the molecules do not allow effective diffusion of the antibody, and thus, it is
found in very low concentration in the intracellular fluids.
Functions of IgM
 IgM is very effective against viruses as less IgM than IgG is enough to neutralize viral infections.
 IgM is also a better agglutinin as it takes 100 to 1000 more molecules of IgG than that of IgM for
the same level of agglutination.
 IgM is involved in activating the classical pathway of complement in the immune system due to

the presence of two Fc regions in close proximity.
Immunoglobulin A (IgA)
IgA or sIgA is the main immunoglobulin found in the mucous membrane in the form of secretory
antibodies. The concentration of IgA is found in small quantities in blood, but it is found in high
concentrations in tears, saliva, and sweat
Structure of IgA
 The molecular size of IgA is 160 kDa with a four-chain monomeric structure, however, it can
occur in dimeric and trimeric forms.
 The heavy chain of IgA is divisible into three constant domains, CH1, CH2, and CH3, and a
variable VH domain.
 The hinge region occurs between the CH1 and CH2 domains held together by disulfide linkages.

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 sIgA has a secretory component as an additional component with a polypeptide chain of 75 kDa
and extracellular proteolytic fragment.
 The molecule also has a J-chain linked to the chains via disulfide bridges. The secretory and J
chain
 facilitates the transport of IgA across epithelial cells and protects the molecule from proteolytic
digestion by enzymes.
Properties of IgA
 IgA is the second most abundant immunoglobulin in

humans, with a concentration of 2-4 mg/ml. It
accounts for about 10-15% of the total serum
concentration but is the most abundant antibody in
external secretions.
 IgA is the first line of defense as it works by

inhibiting bacterial and viral adhesion to epithelial
cells and by neutralizing viral and bacterial toxins
intracellularly.
 The secretory IgA mostly occurs in dimeric form with

two monomeric units linked together by a joining
peptide.
Subclasses of IgA
 IgA has been classified into two subclasses; IgA1 and IgA2.
 IgA1 is the monomeric form, and IgA2 is the dimeric or polymeric form.
 IgA1 occurs in serum IgA (about 80%), which is produced in the bone marrow and released on
the mucosal surfaces.
 The IgA in most locally secreted products is polymeric with a relative release of dimeric IgA2.
 One of the most prominent differences between IgA1 and IgA2 is the hinge region which is quite
extended in IgA1.
Functions of IgA
 IgA is the first line of defense as it protects the body from the entry and colonization of mucosal
surfaces by different foreign particles.
Immunoglobulin D (IgD)

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IgD is a monomeric antibody that occurs on the surface of immature B lymphocytes. It is produced in a
secreted form in a small amount in the blood serum.
Structure of IgD
 IgD has a structural diversity throughout evolution in the vertebrates as it is flexible to

complement the function of IgM.
 It is a glycoprotein with two identical δ heavy chains and two identical light chains.
 IgD found on the surface of B lymphocytes has some extra amino acids at C-terminal in order to
anchor to the membrane.
 The light and heavy chains are linked together by disulfide links, but they have additional
intrachain disulfide links that divide the chains into domains.
 The IgD molecule also has an extended hinge region which increases the flexibility of the
molecule but decreases its resistance against proteolytic cleavage.
Created with BioRender.com
Properties of IgD
 IgD is found in low concentration in serum, and its exact

function in the immune system is not yet clearly
understood.
 It represents about 0.25% of the total serum

immunoglobulins with a relative molecular mass of 185
kDa and a half-life of 2.8 days.
 It also accounts for about 1% of the proteins present in

the plasma membranes of B lymphocytes. Here, it
usually coexpressed with another cell surface antibody,
IgM.
Functions of IgD
 The most important function of IgD is antigen receptor on B cells. It also regulates B cell function
if it encounters an antigen.
 It is also secreted in some amounts in the blood, mucosal secretions, and the surface of innate
immune effector cells.
Immunoglobulin E (IgE)
IgE is a type of immunoglobulin found only in mammals and synthesized by plasma cells. It occurs in a
monomeric form with two ε heavy chains and two light chains.

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Structure of IgE
 IgE has a typical antibody structure with ε heavy chains that have a high carbohydrate content.
 IgE has two identical antigen-binding sites consisting of both light and heavy chains and a
valency of 2.
 Like all antibodies, heavy and light chains are further divided into variable and constant regions.
 The heavy chains consist of five domains, out of which one is variable, and four are constant.
Created with BioRender.com
Functions of IgE
 IgE is mostly associated with allergic

reactions where it binds to
reintroduced antigens and triggers the
release of pharmacologically active
agents.
 It also plays an essential role in

response to allergens and antigen
preparation used in desensitization immunotherapy.
Genetic Basis of Antibody Formation
Key Points
 Virtually all microbes can trigger an antibody response. Successful recognition and eradication of
many different types of microbes requires diversity among antibodies, a result of variation in
amino acid composition that allows them to interact with many different antigens.
 Antibodies obtain their diversity through 2 processes. The first is called V(D)J (variable, diverse,
and joining regions) recombination. During cell maturation, the B cell splices out the DNA of all
but one of the genes from each region and combine the three remaining genes to form one VDJ
segment.
 The second stage of recombination occurs after the B cell is activated by an antigen.In these
rapidly dividing cells, the genes encoding the variable domains of the heavy and light chains
undergo a high rate of point mutation, by a process called somatic hypermutation.
 As a consequence of these processes any daughter B cells will acquire slight amino acid
differences in the variable domains of their antibody chains.This serves to increase the diversity
of the antibody pool and impacts the antibody’s antigen-binding affinity.

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 Point mutations can result in the production of antibodies that have a lower or higher affinity
with their antigen than the original antibody. B cells expressing antibodies with a higher affinity
for the antigen will outcompete those with weaker affinities (called affinity maturation).
Key Terms
 Somatic hypermutation: a cellular mechanism by which the immune system adapts to the new
foreign elements that confront it (for example, microbes). A major component of the process of
affinity maturation, SHM diversifies B cell receptors used to recognize foreign elements
(antigens) and allows the immune system to adapt its response to new threats during the
lifetime of an organism.
 V(D)J recombination: Also known as somatic recombination, this is a mechanism of genetic

recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production
of the immune system.
Virtually all microbes can trigger an antibody response. Successful recognition and eradication of many
different types of microbes requires diversity among antibodies (glycoproteins belonging to the
immunoglobulin superfamily). It is the variety in their amino acid composition that allows them to
interact with many different antigens. It has been estimated that humans generate about 10 billion
different antibodies, each capable of binding a distinct epitope of an antigen. Although a huge repertoire
of different antibodies is generated in a single individual, the number of genes available to make these
proteins is limited by the size of the human genome. Several complex genetic mechanisms have evolved
that allow vertebrate B cells to generate a diverse pool of antibodies from a relatively small number of
antibody genes.
Antibody Structure
Antibodies are typically made of basic structural units—each with two large heavy chains and two small
light chains. There are several different types of antibody heavy chains, and several different kinds of
antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five
different antibody isotypes are known in mammals, which perform different roles, and help direct the
appropriate immune response for each different type of foreign object they encounter. Though the
general structure of all antibodies is very similar, a small region at the tip of the protein is extremely
variable, allowing millions of antibodies with slightly different antigen binding sites to exist. This region is
known as the hypervariable region. Each of these variants can bind to a different antigen. This enormous
diversity of antibodies allows the immune system to recognize an equally wide variety of antigens.
Antibodies obtain their diversity through two processes:
V(D)J Recombination
The first stage is called somatic, or V(D)J, which stands for variable, diverse, and joining regions
recombination. Several sets of genes are located within each of the three regions. During cell
maturation, the B cell will splice out the DNA of all but one of the genes from each region and combine

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the three remaining genes together to form one VDJ segment. This segment, along with a constant
region gene, forms the basis for subsequent antibody production.
It is estimated that given the number of variants in each of the three regions, approximately 10,000-
20,000 unique antibodies are producible. V(D)J recombination takes place in the primary lymphoid
tissue (bone marrow for B cells, and thymus for T cells ) and nearly randomly combines variable, diverse,
and joining gene segments. It is due to this randomness in choosing different genes that it is able to
diversely encode proteins to match antigens.
Figure: Redistribu
tion within the immunoglobulin (antibody) gene: Schematic overview of V(D)J recombination.
Somatic Hypermutation
The second stage of recombination occurs after the B cell is activated by an antigen. In these rapidly
dividing cells, the genes encoding the variable domains of the heavy and light chains undergo a high rate
of point mutation, by a process called somatic hypermutation (SHM). SHM is a cellular mechanism by

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which the immune system adapts to the new foreign elements that confront it and is a major
component of the process of affinity maturation. SHM diversifies B cell receptors used to recognize
antigens and allows the immune system to adapt its response to new threats during the lifetime of an
organism. Somatic hypermutation involves a programmed process of mutation affecting the variable
regions of immunoglobulin genes. SHM results in approximately one nucleotide change per variable
gene, per cell division. As a consequence, any daughter B cells will acquire slight amino acid differences
in the variable domains of their antibody chains. This serves to increase the diversity of the antibody
pool and impacts the antibody’s antigen-binding affinity. Some point mutations will result in the
production of antibodies that have a lower affinity with their antigen than the original antibody, and
some mutations will generate antibodies with a higher affinity. B cells that express higher affinity
antibodies on their surface will receive a strong survival signal during interactions with other cells,
whereas those with lower affinity antibodies will not, and will die by apoptosis. Thus, B cells expressing
antibodies with a higher affinity for the antigen will outcompete those with weaker affinities for function
and survival. The process of generating antibodies with increased binding affinities is called affinity
maturation. Affinity maturation occurs after V(D)J recombination, and is dependent on help from helper
T cells.
Antibody genes also re-organize in a process called class switching, which changes the base of the heavy
chain to another. This creates a different isotype of the antibody while retaining the antigen specific
variable region, thus allowing a single antibody to be used by several different parts of the immune
system.
Theories of antibody formation
There are two sets of theories of antibody formation. These are instructive theory and selective
theories.
Instructive theory
Instructive theory suggests that an immunocompetent cell is capable of synthesizing antibodies of all
specificity. The antigen directs the immunocompetent cell to produce complementary antibodies. Two
instructive theories are postulated as follows:
Direct template theory: This theory was first postulated byBreinl and Haurowitz (1930). They suggested
that a particu-lar antigen or antigenic determinants would serve as a template against which antibodies
would fold. The antibody molecule would thereby assume a configuration complementary to antigen
template.
Indirect template theory: This theory was first postulated byBurnet and Fenner (1949). They suggested
that the entry of anti-genic determinants into the antibody-producing cells induced a heritable change in
these cells. A genocopy of the antigenic determinant was incorporated in genome and transmitted to
the progeny cells. However, this theory that tried to explain specificity and secondary responses is no
longer accepted.
Selective theories

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Three selective theories were postulated as follows:
Side chain theory: This theory was proposed by Ehrlich (1898).According to this theory,
immunocompetent cells have surface receptors that are capable of reacting with antigens, which have
complementary side chains. When antigens are introduced into host, they combine with those cell
receptors that have a complementary fit. This inactivates the receptors. There is an overproduction of
the same type of receptors that circulate as antibodies, as a compensatory mechanism.
Natural selection theory: This theory was proposed by Jerne(1955). According to this theory, during the
embryonic life, mil-lions of globulin molecules were formed against all possible range of antigens. The
antigen when introduced to the host combines selectively with the globulin molecule that has the
nearest comple-mentary fit. The globulin with the combined antigen stimulates antibody-forming cells
to produce the same type of antibody.
Clonal selection theory: Burnet (1957) suggested that immu-nological specificity existed in the cell but
not in the serum and proposed the most acceptable clonal selection theory. According to this theory, a
large number of clones of immunological com-petent cells bearing specific antibody patterns are
produced during fetal development by a process of somatic mutations of immunological competent cells
(ICCs) against all possible antigens.
This theory suggests that an individual ICC expresses mem-brane receptors that are specific for a distinct
antigen. This unique receptor specificity is determined before the lympho-cyte is exposed to antigen.
Binding of antigen to its specific receptor activates the cell and leads to cellular proliferation to form
clones, synthesizing the antibody.
The clonal selection theory is most widely accepted and pro-vides a framework for better understanding
of the specificity, immunological memory, and the property of recognition of self and nonself by
adoptive immunity.
The clonal selection hypothesis has become a widely accepted model for how the immune system
responds to infection and how certain types of B and T lymphocytes are selected for destruction of
specific antigens invading the body.
Figure: A schematic view of clonal selection: Clonal selection of lymphocytes: 1) A hematopoietic stem
cell undergoes differentiation and genetic rearrangement to produce 2) immature lymphocytes with
many different antigen receptors. Those that bind to 3) antigens from the body’s own tissues are
destroyed, while the rest mature into 4) inactive lymphocytes. Most of these will never encounter a
matching 5) foreign antigen, but those that do are activated and produce 6) many clones of themselves.
Four predictions of the clonal selection hypothesis
 Each lymphocyte bears a single type of receptor with a unique specificity (by V(D)J
recombination).
 Receptor occupation is required for cell activation.

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 The differentiated effector cells derived from an activated lymphocyte will bear receptors of
identical specificity as the parental cell.
 Those lymphocytes bearing receptors for self molecules will be deleted at an early stage.
In 1954, Danish immunologist Niels Jerne put forward a hypothesis which stated that there is already a
vast array of lymphocytes in the body prior to any infection. The entrance of an antigen into the body
results in the selection of only one type of lymphocyte to match it and produce a corresponding
antibody to destroy the antigen. This selection of only one type of lymphocyte results in it being cloned
or reproduced by the body extensively to ensure there are enough antibodies produced to inhibit and
prevent infection. Australian immunologist Frank Macfarlane Burnet, with input from David W. Talmage,
worked on this model and was the first to name it “clonal selection theory. ” Burnet explained
immunological memory as the cloning of two types of lymphocyte. One clone acts immediately to
combat infection whilst the other is longer lasting, remaining in the immune system for a long time,
which results in immunity to that antigen. In 1958, Sir Gustav Nossal and Joshua Lederberg showed that
one B cell always produces only one antibody, which was the first evidence for clonal selection theory.
B cells exist as clones. All B cells derive from a particular cell, and as such, the antibodies and their
differentiated progenies can recognize and/or bind the same specific surface
components composed of biological macromolecules ( epitope ) of a
given antigen. Such clonality has important consequences, as
immunogenic memory relies on it. The great diversity in immune
response comes about due to the up to 109 clones with
specificities for recognizing different antigens. Upon
encountering its specific antigen, a single B cell, or a clone of
cells with shared specificity, divides to produce many B cells.
Most of such B cells differentiate into plasma cells that secrete
antibodies into blood that bind the same epitope that elicited proliferation
in the first place. A small minority survives as memory cells that can
recognize only the same epitope. However, with each cycle, the
number of surviving memory cells increases. The increase is
accompanied by affinity maturation which induces the survival of
B cells that bind to the particular antigen with high affinity. This
subsequent amplification with improved specificity of immune response
is known as secondary immune response. B cells that have not been
activated by antigen are known as naive lymphocytes; those that have met
their antigen, become activated, and have differentiated further into fully functional lymphocytes are
known as effector B lymphocytes.
Theories that have been proven correct by clonal selection
Burnet and Peter Medawar collaborated on the study of immunological tolerance, a phenomenon that
can be explained by clonal selection as well as other means. As long as the exposure of cells happens

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early in the organism’s development, it will be tolerated by the organism and will not cause an immune
response to be triggered. There are a large range of lymphocytes seen in the immune system, ranging
from cells that accept self tissue to cells that do not tolerate self tissue. Only cells that are tolerant of
their own tissue endure the embryonic stage, however. The lymphocytes that form as a result of the
introduction of non-self tissue are those that recognise the non-self tissues as belonging to the self.
When Burnet claimed that tissues may be effectively transplanted to foreign recipients under particular
conditions in 1959, the world took notice. This research has resulted in a far better knowledge of the
immune system, as well as significant advancements in the field of tissue transplantation. In 1960,
Burnet and Medawar were jointly awarded the Nobel Prize in Physiology or Medicine.
It was postulated in 1974 by Niels Kaj Jerne, that the immune system is composed of an interconnected
network of cells whose activity is governed by interactions between their variable portions and the
substances they release. In Immune Network Theory, the idea of clonal selection is a fundamental
building block. As a result of his contributions to immunological network theory, Jerne was awarded the
Nobel Prize in Physiology or Medicine in 1984 for his work.
Monoclonal antibodies (Mabs)
 Antibodies are glycoprotein synthesized in blood against specific antigens hust to combat and
give immunity. Such antibodies are heterogenous and are polyclonal antibodies. Therefore they
do not have characteristics of specificity.
 If a specific lymphocyte after isolation and culture invitro becomes capable of producing a single
type of antibody which bears specificity against specific antigen, it is known as monoclonal
antibody.
 These monoclonal antibodies are derived from a single clone of cell which recognize only one
kind of antigen.
 Monoclonal antibodies are produced against a variety of proteins, glycoproteins, glycolipids,

nucleic acids and chemically defined groups linked to protein carriers.
Hybridoma technology for production of monoclonal antibodies:
 Monoclonal antibodies are produced by hybridoma technology.
 The term hybridoma is used to fused cells resulting due to fusion of following two types of cells-
a lymphocytes and tumor cell.
 An antibody producing B- lymphocytes ( eg. Spleen cell of mouse immunized with RBCs
from sheep)

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 A single myeloma cell (eg. Bone marrow tumor cell) that can adopted to grow for
infinite time in culture
 The fused product derived the ability of two different types of cells. ie. Ability to produce large
amount of pure antibodies as lymphocytes and ability to grow or multiply indefinitely like tumor
cell.
Steps in production of monoclonal antibodies:
Step I: Immunization of rabbit or rat and extraction of B-lymphocytes
 In order to isolate B-lymphocyte producing certain antibodies, rabbit or lab rat is immunized
through repeated injection of specific antigen (sheep RBCs)
 A sample of B-cells is extracted from spleen of rabbit or rat
Step II: fusion of myeloma cell with B-lymphocytes:
 The extracted B-lymphocytes is added to a culture of myeloma cell from bone marrow.
 The intended result is the formation of hybridoma cells formed by fusion of B-cell and myeloma
cell.
 The fusion is done by using Polyethylene glycol (PEG) or by electrophoration or by using phages.
Step III: selection of hybridoma cell
 The next step is selection of hybridoma cells.
 The B-lymphocytes contains HPRT1 gene which codes for enzyme Hypoxanthine-guanine
phosphoribosyltransferase (HGPRT). The enzyme HGPRT involved in synthesis of nucleotides
from Hypoxanthine present in culture medium. Therefore B- cells can grow in medium
containing Hypoxanthine amonopterin thymine (HAT media).
 But myeloma cell lack HPRT1 gene so, it does not produce HGPTR enzyme and it does not grow
in HAT medium.
 The myeloma cell fused with another myeloma cell or those do not fused at all die in HAT
medium since they do not utilize Hypoxanthine.
 Similarly, B- cell that fuse with another B- cell or those do not fuse at all die eventually because
they do not have capacity to divide indefinitely,
 So, only hybridoma cell ie. fused cell between myeloma and B-cell can survive and divide in HAT
medium.
 Screening is done to select hybridoma cells which are the desired cell for monoclonal antibodies
production.

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Step IV: culture of Hybridoma cell:
 The selected hybridoma cells are cultured in suitable culture medium, often supplemented with
insulin, transferon, ethanol, amine and other additional hormones.
 Some commonly used culture media for hybridoma cell for production of monoclonal
antibodies are:
 DMEM (Dulbecco’s modified eagle medium)
 IMDM (Iscove’s Modified Dulbecco’s Medium)
 Ham’s F12
 RPMI 1640 medium (Roswell Park Memorial Institute 1640 medium)
Step V: Inoculation of hybridoma cell into suitable host
 These hybridoma cells are then injected into lab animal so that they starts to produce
monoclonal antibodies.
 These hybridoma cells may be frozen and store for future use.
Step VI: extraction and purification of Monoclonal antibodies:
 Monoclonal antibodies from host animal is extracted and purified by one of the following
methods;
 Ion exchange chromatography
 Antigen affinity chromatography
 Radial immunoassay
 Immune precipitation
Application of Monoclonal antibodies:
1. Disease diagnosis:
 ELISA to test HIV, hepatitis, Herpes etc
 RIA- to test viral infection
 Mabs to Hunam chorionic gonadotropin
List of Types of monoclonal antibodies approved for therapeutic application
 Abciximab
 Trade name: Reopro

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 Used after angioplasty to prevent blood clot in coronary artery
 Adalimumab
 Trade name: Humira, Amjevita
 Used to treat rheumatoid arthritis
 Alefacept
 Trade name: Amevive
 Used as immunosuppressive drug
 Alemtuzumab
 Trade name: Campath
 Used for the treatment of chronic lymphocytic leukemia, cutaneous T-cell lymphoma
and T-cell lymphoma
 Basiliximab
 Trade name: Simulect
 Used as immunosuppressive drug
 Belimumab
 Trade name: Benlysta
 Used as immunosuppressive. It inhibits B cell activation.
 Bezlotoxumab
 Trade name: Zinplava
 Used to treatment of recurrence of Clostridium difficile infections
 Canakinumab
 Trade name: (Ilaris
 Used to neutralize Interlukin I beta
 Certolizumab pegol
 Trade name: Cimzia
 Used for the treatment of Crohn’s disease, rheumatoid arthritis, psoriatic arthritis and
ankylosing spondylitis

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 Cetuximab
 Trade name: Erbitux)
 Used for the treatment of metastatic colorectal cancer, metastatic non-small cell lung
cancer and head and neck cancer.
 Daclizumab
 Trade name: Zenapax, Zinbryta
 Used for the treatment of adults with relapsing forms of multiple sclerosis
 Denosumab
 Trade name: Prolia, Xgeva
 Used for the treatment of osteoporosis, treatment-induced bone loss, metastases to

bone, and giant cell tumor of bone.
 Efalizumab
 Trade name: Raptiva
 Used for treatment of autoimmune disease
 Golimumab
 Trade name: Simponi, Simponi Aria
 Inflectra
 Trade name: Remicade
 Ipilimumab
 Trade name: Yervoy
 Used to activate immune system
 Ixekizumab
 Trade name; Taltz
 Used to treat autoimmune disease
 Natalizumab

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 Trade name: Tysabri
 Used for the treatment of multiple sclerosis and Crohn’s disease.
 Nivolumab
 Trade name: Opdivo)
 Used to treat cancer
 Olaratumab
 Trade name: Lartruvo
 Used to treat tumor
 Omalizumab
 Trade name: Xolair)
 Used in treatment of allergies
 Palivizumab
 Trade name: Synagis)
 Used for treatment of Respiratory syncytial virus
 Panitumumab
 Trade name: Vectibix
 Used for colorectal cancer
 Pembrolizumab
 Trade name: Keytruda
 Used in cancer immunotherapy
 Rituximab
 Trade name: Rituxan
 Used to treat autoimmune disease
 Tocilizumab
 Trade name: Actemra
 Used as Immunosupressive drugs
 Trastuzumab

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 Trade name: Herceptin
 Used in treatment of breast cancer
 Secukinumab
 Trade name: Cosentyx
 Used for treatment of psoriasis, ankylosing spondylitis, and psoriatic arthritis.
 Ustekinumab
 Trade name: Stelara
 Used for treatment of Psoriasis and Crohn’s disease
Chimeric Monoclonal Antibody
Definition of Chimeric Monoclonal Antibody
Chimeric antibodies are structural chimeras made by fusing variable regions from one species like a
mouse, with the constant regions from another species such as a human being. Chimerization of
antibodies is a necessary process to reduce immunogenicity and increase serum half-life when preparing
monoclonal antibodies (mAbs) for therapeutic purposes.
Researchers could change the constant regions from one species to another, as well as change from one
subtype to another subtype within the same species. However, making it happen is not as straight and
could be fairly challenging.
Sino Biological has genetically manipulated many antibodies by switching an antibody's constant regions
to meet certain desired features. Our experience covers not only human and mouse antibodies, but also
rabbit, rat, canine antibodies. Our professional scientists are at your service to support your chimeric
antibody production.
Chimeric Antibody Production
Chimeric antibodies can be generated by fairly straightforward genetic engineering, by joining the
immunoglobulin (Ig) variable regions of a selected mouse hybridoma to human Ig constant regions, and
be used as such or as a first stage towards further humanization.
First, the spleen containing B cells from an immunized mouse is collected. The B cells are fused with
myeloma cells to construct hybridoma and isolate a clone producing antigen-specific IgG. The DNA
sequences coding mouse VH and VL are then isolated from the clone, as well as the DNA sequences
coding human immunoglobulin constant regions from human cells. Mouse/human chimeric genes are
constructed by genetic engineering and transfected into mammalian cells. Finally, the clone revealing a
high level expression of chimeric IgG is selected and the IgGs are purified from the culture supernatant.

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Fig 1. How to generate human-mouse chimeric monoclonal antibodies
Chimeric Antibody Applications
Chimeric monoclonal antibodies are very important and powerful for uses in therapeutics and
immunoassays. For in vitro applications such as Immunohistochemistry studies or ELISA assay
development, switching the antibody constant regions to match the species of the host or secondary
antibody could significantly reduce the background staining.
After the FDA approval of rituximab, two additional chimeric antibodies, cetuximab and dinutuximab,
reached the market for oncology indications. Cetuximab was generated by immunizing mice with
purified EGFR and replacing the mouse constant domains of the mouse antibody 225 with those of
human IgG1. The chimeric molecule, named C225, showed around five-fold higher affinity and increased
tumor growth reduction than the parental mouse antibody.
Dinutuximab was developed from a murine antibody specific for GD2. The chimeric molecule (ch14.18),
also a human IgG1, showed identical binding as the murine IgG2a antibody but, the ADCC was 50-fold to
100-fold higher than the parental mouse antibody when using human effector cells. Therefore, in
addition to rendering less immunogenic molecules, chimerization overcame some of the drawbacks of
the early murine monoclonal antibodies by generating therapeutic molecules with the same or
improved affinity than the parental mouse antibodies but with enhanced effector functions.

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UNIT 2 IMMUNE CELLS AND ORGANS
Hematopoiesis: Concept of Haematopoietic stem cells, Process and Regulation
Haematopoiesis
 Haematopoiesis is defined as the process of formation, development and differentiation of

blood cells.
 The blood cells are formed from haematopoietic stem cells (HSCs) which are either multipotent
or pluripotent in nature.
 In the prenatal stage, haematopoiesis occurs in the yolk sac during the first weeks of embryonic
development and transitions to the spleen, liver, lymph nodes and finally in the bone marrow
continuing for lifetime.
Hematopoietic stem cells (HSCs) and types
 Haematopoietic stem cells (HSCs) are special type of cell present in rar the bone marrow, they
are rare and their numbers are strictly controlled by a balance of cell division, death, and
differentiation.
 HSCs divide generating daughter cells. Some daughter cells retain the stem-cell characteristics of
the mother cell having property of self self-renewing and able to give rise to all blood cell types.
While other daughter cells differentiate into progenitor cell that lose their self-renewal capacity
and giving rise to a particular blood cell lineage.
 Therefore, early in hematopoiesis, a multipotent stem cells differentiates along one of two
pathways, giving rise to either a common lymphoid progenitor cells or a common myeloid
progenitor cells
 Both the myeloid and lymphoid lineages are engaged in dendritic cell formation.
 Myeloid cells: It consists of-
 Monocytes
 Eosinophils
 Basophils
 Neutrophils
 Macrophages
 Erythrocytes
 Megakaryocytes

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 Platelets.
 Lymphoid cells: It consists of-
 B cells
 T cells
 Natural killer cells
 Innate lymphoid cells.
The common myeloid progenitor cells:
 The common myeloid progenitor cell (CFU-GEMM) can differentiate into erythrocytes,
thrombocytes, granulocytes, lymphocytes and monocytes.
 For the differentiation and proliferation of CFU-GEMM from HPCs, Interleukin-3 and
Granulocyte-macrophage colony-stimulating factor (GM-CSF) are implicated.
 Stem cell factor (SCF) is a cytokine that can trigger the growth and total number of CFU-GEMM.
 CFU-GEMM is the precursor to several colony forming units which finally give rise to different
blast cells.
 These consists of the following:
 CFU-Meg (from which promegakaryoblasts are produced)
 CFU-E (from which proerythroblasts are produced)
 CFU-GM (both monoblasts and myeloblasts are derived from this type of colony forming
unit)
Process of hematopoiesis:
 According to the monophyletic theory of hematopoiesis, the pluripotent stem cells multiply to
produce more of the pluripotent stem cells, making sure of the steady and lasting supply of
stem cells.
 Some of the pluripotent stem cells now differentiate into precursor cells that are least partially
dedicated to form one type of mature blood cell.
 Pluripotent cells multiply at low pace into one of the five possible unipotential stem cells.
 These unipotential stem cells then multiply rapidly into the precursor of the destined specific
mature blood cell.
 The typical process of hematopoiesis consists of the differentiation of the multipotential

hematopoietic stem cell into either the common myeloid or lymphoid progenitor.

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 Then, relying upon the cytokines and resulting transcription factors that are activated, the
myeloid progenitor can differentiate into a myeloblast.
 This myeloblast leads to granulocyte (basophils, eosinophils, or neutrophils) or monocyte

(macrophages and dendritic cell) development.
 Also, it leads to the differentiation of megakaryocytes into platelets, or erythroblasts into

erythrocytes.
 Lymphoid dendritic cells can form directly from the common lymphoid progenitor.
 Furthermore, the differentiation of the common lymphoid progenitor into a lymphoblast results
the further development of natural killer cells or lymphocytes (T and B cells).
 Once B cells get activated in secondary lymphoid organs, it further differentiate into plasma
cells.
 These plasma cells secrete antibodies.
Regulation of Haematopoiesis:
 Hematopoiesis is largely regulated by the presence of cytokines.
 These cytokines are responsible for regulating the differentiation of multipotential

hematopoietic stem cells into specific cell types by the activation of transcription factors.
 The cytokines is very important for differentiation of particular cell types otherwise animal dies
during embryogenesis.
 Some cytokines that regulates haematopoiesis are:
 Granulocyte macrophage-colony stimulating factor (GM-CSF):
 It enhances the myeloid lineage, finally leading to the differentiation of

granulocytes and macrophages. Such cytokines are termed as growth factors.
 These growth factors are needed throughout the process of hematopoiesis

functioning in order to activate transcription factors.
 Transcription factor GATA-2:
 It is required for the development of all hematopoietic lineages; in its absence

animals die during embryogenesis.
 Transcriptional regulator Bmi-1:
 It is required for the self-renewal of HSCs, and in its absence animals die within
2 months of birth because of the failure to repopulate their red and white
blood.

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 Other Examples of cytokines involved in haematopoiesis are:
 Colony-stimulating factors (CSFs)
 Erythropoietin (EPO)
 Thrombopoietin (TPO)
 Granulocyte colony-stimulating factor (G-CSF)
 Monocyte colony-stimulating factor (M-CSF)
 Tumor necrosis factor (TNF)
 Transforming growth factor (TGF)
 Stem cell factor (SCF)
 Leukemia inhibitory factor (LIF)
Erythropoiesis:
 The process of formation of red blood cells termed as erythrocytes is known as erythropoiesis.
 It is enhanced by decreased levels of oxygen in the blood, which signals for the secretion of
erythropoietin.
 Erythropoietin is a hormone central to the formation of red blood cells.
 Erythropoiesis takes on average 2 days to be completed from to form mature red blood cell
from unipotential hematopoietic cell.
 2 million erythrocytes are produced every second in our bodies.
 Hematopoietic cells determined to become red blood cells usually get smaller and more
condensed as they mature until there is finally loss of their nuclei.
 The unipotential cell becomes proerythroblast, which has uncondensed nucleus and has
basophilic or blue cytoplasm.
 Then the cell becomes a basophilic erythroblast, which is followed by a polychromatophilic

erythroblast stage.
 In polychromatophilic erythroblast stage, the nucleus becomes more condensed than the latter
two stages and the cytoplasm is reduced.
 In the succeeding orthochromatophilic erythroblast stage, the nucleus is much smaller than that
of the prior stages having a pinker cytoplasm.

MICROBIOLOGY
 Then comes the reticulocyte stage, where the red blood cell lacks nucleus, but still stains
somewhat blue because of the remnants of polyribosomes within the cell.
 Ultimately, the erythrocyte is the mature red blood cell, with no nucleus and no polyribosome
remnants and as a result stains pink.
Granulopoiesis
 The process of formation of granulocytes is termed as granulopoiesis.
 Granulocytes are white blood cells having multi-lobular nuclei and cytoplasmic granules.
 The unipotential hematopoietic cell which forms a myeloblast is large.
 It has a cytoplasm that stains blue with a large nucleus.
 This cell gives rise into a promyelocyte that contains azurophilic granules. Then it becomes a
myelocyte, which has a non-indented still rather large nucleus.
 This cell then gives rise to a metamyelocyte, which is alike in size to a mature granulocyte and
the nucleus starts to become indented.
 After this stage is the band cell stage, where the nucleus resembles a horseshoe and has
definitive indentation.
 Ultimately, there is the mature granulocytes having a lobed nucleus and cytoplasmic granules.
 The entire process occurs over a period of 2 weeks.
Monopoiesis:
 The process by which monocytes are formed is termed as monopoiesis.
 The monoblast is the committed progenitor cell, found only in the bone marrow. Also,
monoblast has a basophilic cytoplasm without granules.
 These monoblasts give rise to promonocytes, which are smaller in size with nuclei that become
slightly indented, before becoming monocytes.
 Monocytes have kidney-shaped nuclei and can develop into dendritic cells or macrophages.
Lymphopoiesis:
 The formation of lymphocytes, starts from their first committed progenitor cells, lymphoblasts,
this process is called Lymphopoiesis.
 These cells after maturation, are able to differentiate into either B, T or natural killer cells.
Thrombopoiesis:

MICROBIOLOGY
 Megakaryocytes, which are extremely large cells within the bone marrow forms the platelets,
this process is termed as thrombopoiesis.
 When the plasma membranes of megakaryocytes are fragmented, the origin of individual
platelets take place, thus generating platelets containing many granules.
Hematopoiesis: Types of Haematopoietic stem cells, Process and Regulation

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Immune Cells and Organs
The immune system is made up of different immune organs and tissues located all over the body. The
immune organs are categorized based on their functions and there are two main categories, which
include:
1. Primary lymphoid organs provide a development and maturation site for lymphocytes, and
2. Secondary lymphoid organs whose function includes trapping antigens from the tissues, and the
vascular spaces. They are also the site for lymphocyte interaction with the antigens.
All these organs are connected by the lymphatic system and the blood vessels into a functional unit. In
the blood and the lymph and populating the lymphoid organs are various types of white blood cells
(leukocytes) that play a key role in the body’s immune responses, therefore defining the cells of the
immune system. Nevertheless, white blood cells are an assemblage of different immune cells. White
blood cells provide the defense mechanisms of the body fighting off foreign elements (antigens) from
the body. Under the White Blood Cells group of cells, they can be categorized into lymphocytes,
(including T-lymphocytes, B-lymphocytes, and Natural Killers cells), neutrophils, monocytes, and
macrophages.
In all these categories, only the lymphocytes have the characteristics of diversity, specificity, memory,
and self/nonself recognition, which are the hallmark features of the adaptive immune responses. All the
other cells play additional roles in adaptive immunity such as activation of lymphocytes, increasing the
effector mechanisms of antigen clearance by phagocytosis, or secreting various immune-effector
molecules. Some white blood cells secrete protein molecules known as cytokines which are
immunoregulators (regulate the immune responses). Other major proteins of the immune system
include antibodies produced by B-lymphocytes, and complement proteins (activated by antibodies).
Types of Cells of Immune System
The lymphocytes are the central

cells of the immune system
which are responsible for
adaptive immunity and
immunological features of
diversity, specificity, memory,
and self/non-self recognition.

MICROBIOLOGY
The other immune cells function to engulf and destroy micro-organisms, present antigens and secrete
cytokines.
Lymphoid cells (Lymphocytes)
The lymphocytes make up 20%–40% of the body’s white blood cells and 99% of the cells in the lymph.
There are about 1011 lymphocytes in the human body. These lymphocytes circulate continuously in the
blood and the lymph hence they are able to migrate into the body tissue spaces and lymphoid organs,
therefore integrating the immune system to a high degree.
Types of Lymphocytes
The lymphocytes
are broadly
divided into three
populations based
on their functions
and cell-
membrane
components i.e:
1. B-lymphocytes
 B-
lymphocytes are also known as B-cells and on lab reports, they are known as CD19 or CD20 cells.
 They are the specialized cells of the immune system whose major function is to produce
antibodies also known as immunoglobulins or gamma globulins.
 B-lymphocytes are synthesized and mature in the bone marrow from the hematopoietic stem
cells, and after which they mature, migrate, and express themselves by forming unique antigen-
binding receptors on their membranes, known as B-cell receptors or antibodies.
 Migration of mature B-cells moves to the bone marrow, lymph nodes, spleen, some parts of the
intestines, and the bloodstream.

MICROBIOLOGY
B-Cells
 When a naive B-cell interacts with an antigen for the first time and it has to match membrane-
bound receptors (antibodies), the antibodies bound to the B-cell bind the antigen causing the B-
cell to divide rapidly, and its progenitors to differentiate into memory B-cells and effector B-
cells known as plasma cells.
 The Memory B-cells have a long life span than the naive cells, expressing the same membrane-
bound antibody as the parent B-cells.
 The plasma cells are responsible for producing the antibodies that can be secreted into the
bloodstream, tissues, respiratory secretions, intestinal secretions, and tears.
 Therefore, antibodies are highly specialized serum protein molecules.
 The plasma cells have a short life span of few days but they secrete large amounts of antibodies
during this time, with approximately 2000 molecules of antibodies per plasma cell per second.
 The secreted antibodies play the major effector roles in the humoral immune responses.
 Note that, during maturation, B-cells are trained not to produce antibodies on healthy tissues.
 The antibody molecules are specifically designed for every foreign antigen they encounter and
interact like a lock and key mechanism.
 Therefore B-cells have the ability to produce vitally a variety of antibodies for all microbes in our
environment, however as stated above, each plasma cell produces only one kind of antibody.
 Antibodies’ varieties are based on their specialized functions in the body with variations in their
chemical structure, which ultimately determine the class of antibody.
2. T-Lymphocytes
 T-lymphocytes are also known as T-cells, often named in lab reports as CD3 cells

MICROBIOLOGY
 They also arise in the bone marrow but migrate to the thymus gland for maturation, where they
express a unique antigen-binding molecule on its membrane known as the T-cell receptor.
 The name T originated from its site of maturation, the Thymus.
 Mature T-cells leave the thymus and populate other organs of the immune system, such as the
spleen, lymph nodes, bone marrow, and blood.
 Unlike the B-cell receptors that can recognize antigens alone, T-cell receptors only recognize
antigens that are bound to cell membrane proteins known as Major Histocompatibility Complex
(MHC) molecules.
 The MHC molecule recognizes antigens that are presented to them by antigen-processing cells
(APCs) on their cell membrane.
 The two major classes of MHC molecules are Class I MHC molecules, which are expressed by
nearly all nucleated cells of vertebrate species, consist of a heavy chain linked to a small
invariant protein called 2-microglobulin. Class II MHC molecules, which consist of an alpha and a
beta glycoprotein chain, are expressed only by antigen-presenting cells.
 When a naive T cell encounters an antigen combined with an MHC molecule on a cell, the T cell
proliferates and differentiates into memory T cells and various effector T cells.
T-Cells
 The T-cells are classified into three categories: T helper (Th), T cytotoxic (Tc), and T suppressor
(Ts) cells.
 The Th and Tc cells are differentiated from each other with the presence of their CD4 and CD8
membrane glycoproteins on their surfaces.
 T cells naturally displaying CD4 function as T helper (Th) cells while those displaying CD8

naturally function as T cytotoxic (Tc) cells.
 The Th cells recognize and interact with antigens that are presented on the MHC class II
molecule complex, then they become activated becoming effector cells that are able to secrete
various growth factors that are collectively known as cytokines.

MICROBIOLOGY
 The cytokines that are secreted are actively involved in the activation of B-cells, T-cytotoxic cells,
macrophages, and other immune cells.
 The cytokine patterns produced by the activated Th cells result in different immune responses.
The Th-derived cytokines enable the recognition of an antigen-MHC class I molecule complex by
the Tc cells which then proliferate and differentiate into effector cells known as Cytotoxic T-
lymphocytes (CTL).
 The T-cytotoxic cells have the ability to induce cytokine secretion, unlike the Cytotoxic T-Cells
which do not induce secretion of cytokines, rather they exhibit cell-killing or cytotoxic activity.
 Cytotoxic T-lymphocytes (CTL) play a key role in monitoring the body cells and eliminating any of
these cells that display antigens such as tumor cells, cells infected with viruses, and cells of a
foreign tissue graft.
 CTLs target foreign antigen (altered self-cells) complexes displayed by the class I MHC molecule.
3. Natural killer cells (NK cells)
 These are large granular lymphocytes, that do not express surface markers like the B and T-cell
lineages
 They were first described in 1976 by indications of the presence of a small population of large
granular lymphocytes that had a cytotoxic effect against a wide range of tumor cells in the
absence of any previous immunization with the tumor.
 These cells also indicated that they play key roles in host defense against tumor cells and cells
infected with some, not all viruses.
 They constitute 5-10% of lymphocytes in the human peripheral blood.
Natural Killer Cell
 Their ability to recognize antigens is based on two

mechanisms:
 They can employ NK cell receptors to distinguish

abnormalities such as a reduction in the expression of
class I MHC molecules and the abnormal profile of the
surface antigens that are displayed by some tumor
cells and cells infected by some viruses.
 Secondly, the NK cells also recognize potential target

cells which are tumor cells and cells that are infected
by viruses. These target cells display antigens against which the immune system has
already produced antibody response to as antitumor or antiviral antibodies, that bind to
the surfaces of these targets.

MICROBIOLOGY
 The NK cells express membrane receptors called CD16, which are receptors for the carboxyl-
terminal end of the IgG molecule, Fc region. The NK CD16 receptors attach to these antibodies
and destroy the targeted cells subsequently, by a mechanism known as the Antibody-
dependent cell-mediated cytotoxicity (ADCC).
 Therefore, NK cells play an important role in host defense mechanisms against tumors.
 For example, in humans, the Chediak-Higashi syndrome, an autosomal recessive disorder is

associated with impairment in neutrophils, macrophages, and NK cells and an increased
incidence of lymphomas. Likewise, mice with an autosomal mutation called beige lack NK cells;
these mutations are more susceptible to tumor growth than normal mice following injection
with live tumor cells.
 There are some unique NK cells known as the NK1-T-cells which have been recognized to have
some combined characteristics of the T-lymphocytes and the Natural Killer cells. They have T-
cell receptors (TCRs) that interact with the MHC-like molecule known as a CD1, unlike the
normal TCRs of the T-cell which interact with class I or class II MHC molecules. Additionally, like
the NK cells, they have variable levels of CD16 and other NK receptors which enable it to kill
cells.
 A population that is triggered by NK1-T cells secretes large amounts of cytokines, rapidly. These
cytokines support the antibody production by B-cells, also inflammation, and the development
and expansion of cytotoxic T-cells.
 Some immunologists view this cell type as a kind of rapid response system that has evolved to
provide early help while conventional TH responses are still developing.
Mononuclear Phagocytes
 These are immune cells i.e monocytes that are freely circulating in blood and macrophages that
are found in the tissues.
 During hematopoiesis in the bone marrow, granulocyte-monocyte progenitor cells differentiate

into promonocytes, which leave the bone marrow and enter the blood, where they differentiate
further into mature monocytes.
 Monocytes circulate in the bloodstream for about 8 h, during which they enlarge and then
migrate into the tissues and differentiate into specific tissue macrophages or into dendritic cells.
 Differentiation of monocyte into a tissue macrophage involves a number of changes
 The cell enlarges five- to tenfold
 Its intracellular organelles increase in both number and complexity
 It acquires increased phagocytic ability and produces higher levels of hydrolytic enzymes

MICROBIOLOGY
 It begins to secrete a variety of soluble factors.
 Macrophages are dispersed throughout the body. Some take up residence in particular tissues,
becoming fixed macrophages, whereas others remain motile and are called free, or wandering,
macrophages.
 Free macrophages travel by amoeboid movement throughout the tissues. Macrophage-like cells
serve different functions in different tissues and are named according to their tissue location:
 Alveolar macrophages in the lung
 Histiocytes in connective tissues
 Kupffer cells in the liver
 Mesangial cells in the kidney
 Microglial cells in the brain
 Osteoclasts in bone
 Macrophages are normally in a resting phase but they can be activated by several immune
responses.
 For example, the phagocytic mechanism of certain antigens is normally the initial stimulus for
macrophages. However, macrophage activity can be further enhanced by cytokines secreted by
activated TH cells, by mediators of the inflammatory response, and by components of bacterial
cell walls.
 One of the most potent activators of macrophages is interferon-gamma which is secreted by

activated TH cells.
 Activated macrophages effectively eliminate potential pathogens than the resting macrophages
because they exhibit greater phagocytic activity, an increased ability to kill ingested microbes,
increased secretion of inflammatory mediators, and an increased ability to activate T cells.
 Additionally, activated macrophages, but not resting ones, secrete various cytotoxic proteins
that help them eliminate a broad range of
pathogens, including virus-infected cells, tumor cells, and intracellular bacteria.
 Activated macrophages also express higher levels of class II MHC molecules, allowing them to
function more effectively as antigen-presenting cells. Thus, macrophages and TH cells facilitate
each other’s activation during the immune response.
 Some of the functions of macrophages include:
 Phagocytosis -Phagocytosis of bacteria, viruses, and other foreign particles is the most
important function of macrophages. The macrophages on their cell surfaces have Fc

MICROBIOLOGY
receptors that interact with the Fc component of the IgG, thereby facilitating the
ingestion of the opsonized organisms. They also have receptors for C3b, another
important opsonin. After ingestion, the phagosome containing the microbe fuses with a
lysosome. The microbe within the phagolysosome is killed by reactive oxygen, reactive
nitrogen compounds, and lysosomal enzymes.
 Antimicrobial and cytotoxic activities include the oxygen-dependent and oxygen-

independent cytotoxicity/killing.
 Antigen processing – After ingestion and degradation of foreign materials, the fragments
of antigen are presented on the macrophage cell surface in conjunction with class II
MHC proteins for interaction with the TCR of CD4+ helper T cells. Degradation of the
foreign protein is stopped following the association of antigen with the class II MHC
proteins in the cytoplasm. This is followed by transportation of the complex to the cell
surface by transporter proteins.
 Secretion of growth factors important for the development of an immune response such
as cytokines, such as interleukin 1 (IL-1), TNF-α, and interleukin 6 (IL-6), that promote
inflammatory responses, complement proteins, hydrolytic enzymes, and a cascade of
Tumor Necrotic Factors, TNF-α (GM-CSF, G-CSF, M-CSF) that induce and kill tumor cells
and promote hematopoiesis.
Granulocytic Cells
 Granulocytes are white blood cells (leukocytes).
 They are classified based on their cellular morphologies and cytoplasmic staining characteristics
and they include neutrophils, eosinophils, basophils, or mast cells.
 All granulocytes have multilobed nuclei that make them visually distinctive and easily
distinguishable from lymphocytes, whose nuclei are round. The cytoplasm of all granulocytes is
replete with granules that are released in response to contact with pathogens.
 These granules contain a variety of proteins with distinct functions: Some damage pathogens
directly; some regulate trafficking and activity of other white blood cells, including lymphocytes;
and some contribute to the remodeling of tissues at the site of infection.
 Neutrophils have a multilobed nucleus and a granulated cytoplasm that stains with both acid
and basic dyes; it is often called a polymorphonuclear leukocyte (PMN) for its multilobed
nucleus.
 The eosinophils have a bilobed nucleus and a granulated cytoplasm that stains with the acid dye
eosin red (hence its name).
 The basophil has a lobed nucleus and heavily granulated cytoplasm that stains with the basic
dye methylene blue.

MICROBIOLOGY
 Both neutrophils and eosinophils are phagocytic, whereas basophils are not.
 Neutrophils constitute the majority (50% to 70%) of circulating leukocytes and are much more
numerous than eosinophils (1%–3%), basophils (≤ 1%), or mast cells (≤ 1%).
Neutrophils
 Neutrophils are produced by hematopoiesis n the bone marrow. They are released into the
peripheral blood and circulate for 7–10 h before migrating into the tissues, where they have a
life span of only a few days.
 In the bone marrow, a surmountable level of neutrophils is produced in response to the types of
infections and they are normally the first cells that arrive at the site of inflammation.
 The resulting transitory increases in the number of circulating neutrophils known as

leukocytosis, which is an indicator of an infection, medically.
 The movement of circulating neutrophils into tissues is also known as extravasation.
 Extravasation takes place in several steps:
 Adherence to the vascular endothelium
 Penetration into the gap between adjacent endothelial cells lining the vessel wall
 Penetration into the vascular basement membrane and moving out into the tissue
spaces.
 Several substances can be generated during an inflammatory reaction which serves as

chemotactic factors. They promote the accumulation of neutrophils at the site of inflammation.
Some of these chemotactic factors include complement components, blood-clotting system
components, and several cytokines secreted by activated Th Cells and macrophages.
 The functions include:
 Neutrophils are also active phagocytes just like macrophages and the mechanism of
phagocytosis is similar to that of macrophages except for the lytic enzymes and
bactericidal substances in neutrophils which are contained within primary and
secondary granules.
 The neutrophils have larger denser primary granules which are a type of lysosome
containing peroxidase, lysozyme, and various hydrolytic enzymes, and smaller
secondary granules that contain collagenase, lactoferrin, and lysozyme.
 Both the primary and the secondary granules fuse with the phagosomes and digest and
eliminate the contents similar to macrophages.

MICROBIOLOGY
 Neutrophils also employ both oxygen-dependent and oxygen-independent pathways to
generate antimicrobial substances.
 Neutrophils exhibit a larger respiratory burst than macrophages and they are able to
generate more reactive oxygen intermediates and reactive nitrogen intermediates.
 Neutrophils also express higher levels of defensins than macrophages.
Eosinophils
 They are motile phagocytic cells that can migrate from the blood into the tissue spaces.
 They have a phagocytic mechanism of eliminating antigens but their role as phagocytic cells is
much less significant than that of neutrophils.
 They play a role in defense against multicellular parasitic organisms including worms.
 The secreted contents of eosinophilic granules may damage the parasite membrane. They can
be found clustering around invading worms, whose membranes are damaged by the activity of
proteins released from eosinophilic granules. Like neutrophils and basophils, eosinophils may
also secrete cytokines that regulate B and T lymphocytes, thereby influencing the adaptive
immune response.
 In areas where parasites are less of a health problem, eosinophils are better appreciated as
contributors to asthma and allergy symptoms.
Basophils
 Basophils are nonphagocytic granulocytes containing large granules that are filled with
basophilic proteins that stain blue in standard H & E staining methodologies.
 Naturally, basophils are in the body’s normal circulation but they can be very potent.
 They function by binding to circulating antibodies and react by the content of their granules
which are pharmacologically active substances found in their cytoplasm.
 These substances play a major role in certain allergic responses. For example, histamines are the
most common and well-known protein in that basophilic granules. They play a role in increasing
blood vessel permeability and smooth muscle activity.
 Additionally, just like the eosinophils, basophils are also crucial in response to parasites, and
particularly the helminths (worms).
 Basophils also secrete cytokines that assist in the modulation of the adaptive immune response.

MICROBIOLOGY
Mast Cells
 Mast cells are formed in the bone marrow.
 They are released from the bone marrow into the

blood as undifferentiated cells, and when they enter
the tissues they then mature.
 Mast cells can be found in a wide variety of tissues,

including the skin, connective tissues of various
organs, and mucosal epithelial tissue of the
respiratory, genitourinary, and digestive tracts.
Mast Cell
 Like circulating basophils, these cells have large

numbers of cytoplasmic granules that contain
histamine and other pharmacologically active substances.
 Mast cells also play an important role in the development of allergies.
 Basophils and mast cells share many characteristics however, their relationship is not
unequivocally understood. Some speculations state that basophils are the blood-borne version
of mast cells; others speculate that they have distinct origins and functions.
Dendritic Cells
 These are special cells that were discovered by Ralph Steinman in the mid-1970s, and he won a
noble prize in 2011 for this discovery.
 The dendritic cells acquire their name because they are covered with long membrane extensions
resembling the dendrites of the nerve cells.
 Their membranous extension extends and retracts dynamically, increasing the surface area
available for browsing lymphocytes.
 They are very diverse according to research, and they seem to arise from both the myeloid and
lymphoid lineages of hematopoietic cells.
 They are not easily isolated by conventional methods because the cell isolation damages their
long extensions.
 Dendritic cells generally perform the distinct functions of antigen capture in one location and
antigen presentation in another.
 Outside lymph nodes, immature dendritic cells monitor the body for signs of invasion by
pathogens and capture intruding or foreign antigens.

MICROBIOLOGY
 They then process these antigens, then migrate to lymph nodes, where they present the antigen
to naïve T cells, initiating the adaptive immune response.
 When acting as guards in the periphery, immature dendritic cells take on their cargo of antigen
in three ways.
 engulf it by phagocytosis
 internalize it by receptor-mediated endocytosis
 absorb it by pinocytosis.
 During the maturation process though, they shift from an antigen-capturing phenotype to one
that is specialized for the presentation of
antigen to T cells. When transitioning, some attributes are lost and others are gained. Lost is the
capacity for phagocytosis and large-scale pinocytosis.
 However, the ability to present antigen increases significantly, as does the expression of
costimulatory molecules that are essential for the activation of naïve T cells.
 After activation, dendritic cells abandon residency in peripheral tissues, enter the blood or
lymphatic circulation, and migrate to regions of the lymphoid organs, where T cells reside, and
present antigen.
 There are many types of dendritic cells, although most mature dendritic cells have the same
major function, the presentation of antigen to TH cells.
 There are four types of dendritic cells known:
 Langerhans cells
 interstitial dendritic cells
 myeloid cells
 lymphoid dendritic cells.
 Each of these types arises from hematopoietic stem cells via different pathways and in different
locations.
 However different, they all constitutively express high levels of both class II MHC molecules and
members of the co-stimulatory B7 family.
 Therefore, they are representatively more potent antigen-presenting cells than macrophages
and B cells, both of which need to be activated before they can function as antigen-presenting
cells (APCs).

MICROBIOLOGY
 Immature or precursor forms of each of these types of dendritic cells acquire antigen by
phagocytosis or endocytosis; the antigen is processed, and mature dendritic cells present it to
TH cells.
 Following microbial invasion or during inflammation, mature and immature forms of Langerhans
cells and interstitial dendritic cells migrate into draining lymph nodes, where they make the
critical presentation of antigen to TH cells that is required for the initiation of responses by
those key cells.
 Another type of dendritic cell, the follicular dendritic cell, does not arise in the bone marrow
and has a different function from the antigen-presenting dendritic cells.
 Follicular dendritic cells do not express class II MHC molecules and therefore do not function as
antigen-presenting cells for TH-cell activation.
 These follicular dendritic cells were named for their exclusive location in organized structures of
the lymph node called lymph follicles, which are rich in B cells. Although they do not express
class II molecules, follicular dendritic cells express high levels of membrane receptors for
antibody, which allows the binding of antigen-antibody complexes.
 The interaction of B cells with this bound antigen can have important effects on B cell responses.
Organs of the Immune System
An extensive network of tissues, organs,

proteins, and cells make up the body’s
immune system.
An immune system working correctly can

distinguish healthy tissue and foreign
objects. It will initiate a complex attack to
defend the body from invaders if it
identifies an unwanted element. Also, it
detects and removes damaged and dead
cells.
But, the immune system is not always

reliable. For example, when a person has
a medical condition or needs medications
that alter how the system functions, it may be unable to fight efficiently.
In autoimmune disorders and allergies, the immune system misinterprets healthy tissue and starts an
unwanted attack, resulting in painful and life-threatening symptoms.

MICROBIOLOGY
In this article, we will examine organs of the immune system and how they function to protect the body
from diseases and other outside invaders.
An Overview of Immune System
The immune system functions like a police force. It wanders the entire area and alerts for assistance
when it notices a disturbance. It differs from other systems in this way because it must be able to
respond in any area of the body. Innate immunity and adaptive immunity are the two layers of defence
offered by the immune system.
In general, exposure to many pathogens strengthens the immune system. By adulthood, most people
will have been exposed to several pathogens and strengthened their immune systems.
When the body creates an antibody, it stores a copy so that it can respond more quickly the next time
the same antigen surfaces.
The immune system is composed of two basic parts:
1. The innate immune system: Individuals are born with this immune system.
2. The adaptive immune system: Individuals develop this immunity when exposed to organisms or
chemicals released by microorganisms.
The Innate Immune System
People have some immunity from birth, ready to fight against invaders immediately.
The external barriers of our body, which serve as the first line of defence against pathogens, such as the
skin and gut and throat mucous membranes, are a part of our innate immunity.
This response is non-specific and generic.
Macrophages will engage in combat with infections if they can evade the innate immune system. In
addition, cytokines produced by macrophages help the body respond to inflammation.
The Adaptive (Acquired) Immune System
The ability to defend against infections improves over time.
The body produces a variety of antibodies to various pathogens due to immunisations and exposure to
various diseases. Immunological memory is a term used by
doctors to describe this condition when the immune system
recalls earlier enemies.
Active Immunity

MICROBIOLOGY
Active immunity involves the body’s immediate reaction to a foreign antigen. The body keeps track of
the infections it has already encountered in the acquired or adaptive immune system. This is a direct
effect of the active immune system.
We develop active immunity when we contact the pathogen or its antigen. When a pathogen enters the
body for the first time, some antibodies that combat it are stored in case the pathogen attacks again.
This is referred to as natural active immunity.
Passive Immunity
Antibodies acquired from outside the body trigger an immunological response known as passive
immunity. The first encounter with a disease is always a little tough on the body because the body’s
initial response to it is relatively weak.
This sort of immunity is temporary and comes from a third party. For instance, a newborn acquires
antibodies from their mother through the placenta before birth and breast milk after birth.
This passive immunity protects infants from several infections throughout their early years.
Organs of the Immune System
The thymus and bone marrow are important primary lymphoid organs. Secondary lymphatic tissues like
the spleen, lymph arteries, tonsils, lymph nodes, skin, adenoids, and liver are all important parts of the
immune system.
Primary Lymphoid Organs
The primary sites of lymphocyte development, or lymphopoiesis, are known as primary lymphoid organs
(PLO). Lymphocytes develop from lymphoid stem cells, multiply, and mature into useful cells known as
immuno-competent cells. B-cell maturation occurs in the bone marrow of animals, whereas T-cell
maturation occurs in the thymus.
Thymus
This tiny organ, located in the upper chest below the breast bone, helps to mature a particular type of
white blood cell. The particular task of this cell is to recognise and memorise an intruder so that a
counter-attack can be easily mounted the next time this intruder attacks.
The thymus provides an inductive environment for forming T lymphocytes from haematopoietic stem
cells. Thymic stromal cells also enable the selection of a useful and self-tolerant T cell repertoire. As a
result, the thymus’ induction of central tolerance is one of its most significant functions.
Bone Marrow
Red blood cells, plasma cells, several types of white blood cells, and other immune cells are all produced
from stem cells found in the spongy interior of the bones. Every day, the bone marrow produces billions
of new blood cells and releases them into the blood.

MICROBIOLOGY
The primary site of B-cell maturation and propagation in adults is the bone marrow, where all other
circulating blood cells are produced. The process of producing all blood cells throughout foetal
development is known as haematopoiesis. It first occurs in the blood island of the yolk sac and para-
aortic mesenchyme, followed by the liver and spleen.
Secondary Lymphoid Organs
In addition to the primary lymphoid organs, there are a few other lymphoid organs known as secondary
lymphoid organs. The spleen and lymph nodes are the two most significant and well-organised
secondary lymphoid organs.
The secondary (or peripheral) lymphoid organs (SLO) sustain and initiate an adaptive immune response.
Antigen-induced lymphocyte activation takes place in the secondary lymphoid organs. Until they come
into contact with their particular antigen, mature lymphocytes travel back and forth between the blood
and secondary lymphoid organs. Clonal growth and affinity maturation are produced by activation.
Lymph Nodes
The network of lymphatic channels (also known as lymphatic vessels) and lymph nodes are connected by
lymphatic nodes.
Immune cells found in lymph nodes examine foreign pathogens introduced into the body. These tiny
glands filter and kill them to prevent germs from spreading to other body areas. They are a component
of the lymphatic system in our body. The individual lymphocytes (white blood cells) are then activated,
replicated, and sent to combat that specific invader.
Numerous lymph nodes can be found throughout the body, especially in the groin, armpits, and neck.
Lymph nodes that are swollen and painful are a sign that the body is fighting infections.
Spleen
The secondary lymphoid organ, the spleen, is situated high in the left abdominal area. Spleens are
designed to filter blood, capture blood-borne antigens, and react to systemic infections.
The primary functions of the spleen are:
 Production of immune cells to fight antigens
 Removal of particulate matter and aged blood cells (red blood cells)
 Production of blood cells during foetal life.
The spleen has primarily efferent lymphatic channels, similar to the thymus. It receives blood from both
the splenic artery and the short gastric arteries. The spleen produces antibodies in its white pulp and
eliminates antibody-coated blood cells and germs through lymph nodes and blood circulation.

MICROBIOLOGY
Mucosal-Associated Lymphoid Tissue
Along with lymph nodes, the spleen’s mucosal-associated lymphoid tissue (MALT) is also regarded as a
secondary lymphoid organ. Lymphoid tissues on a thin layer surround the mucous membranes of the
urogenital, respiratory, and digestive systems.
MALTs are designed to produce massive, antibody-producing plasma cells, which are essential for the
body’s defence mechanism.
Lymphatic Vessels
The lymphatic vessels, also known as lymph vessels, are tubes with thin walls transporting lymph
between various body parts. They consist of the tubular lymph capillary vessels, the larger collecting
vessels, the right lymphatic duct, and the left lymphatic duct (thoracic duct).
Interstitial fluid in the tissues is mostly absorbed by lymph capillaries, which then move the fluid into
larger collecting ducts, where it is ultimately returned to the bloodstream via a subclavian vein.
Other Lymphoid Tissues
To protect the body against infections and the growth of tumours, lymphoid tissue connected to the
lymphatic system performs immunological activities. It is made up of connective tissue composed of
reticular fibres (that have a variety of leukocytes or white blood cells, primarily lymphocytes) through
which the lymph passes. Lymphoid follicles are areas of lymphoid tissue that are densely filled with
lymphocytes.
UNIT 3 : MAJOR HISTOCOMPATIBILITY COMPLEX AND

ANTIGEN PRESENTATION
MHC Molecules- Definition, Properties, Class, Types, Pathways
The major histocompatibility complex can be defined as a tightly linked cluster of genes whose products
play an important role in intercellular recognition and in discrimination between self and non-self. The
term ‘histo’ stands for tissue and ‘compatibility’ refers to ‘getting along or agreeable’. On the other
hand, the term ‘complex’ refers to the ‘genes that are localized to a large genetic region containing
multiple loci’. These genes code for antigens which involve the determination of the compatibility of the
transplanted tissue. The compatible tissues will be accepted by the immune system while the histo-
incompatible ones are rejected. The rejection of foreign tissue leads to an immune response to cell
surface molecules. The concept was first identified by Peter Gorer and George Snell. The main function
of MHC molecules is to bring antigen to the cell surface for recognition by T cells. In humans, the genes
coding for MHC molecules is found in the short arm of chromosome 6.
Major Histocompatibility Complex (MHC) Molecules Characteristics

MICROBIOLOGY
 The Major Histocompatibility complex is a genetic locus that encodes the glycoprotein
molecules (transplantation antigens) which are responsible for tissue rejection of grafts between
genetically unidentical individuals.
 It is also the molecule that binds the peptide antigens processed by Antigen-presenting Cells and
presents them to T-cells, hence they are responsible for antigen recognition by the T-cell
receptors.
 Unlike the B-cell receptors that directly interact with the antigens, the T-cell receptors have an
intertwined relationship with the MHC molecule, in that T-cell receptors can only receive and
bind processed antigens in form of peptides that are bound to the MHC molecule, and
therefore, T-cell receptors are specific for MHC molecules.
 In humans, the Major Histocompatibility complex is known as Human Leukocyte Antigen (HLA).
There are three common MHC molecules i.e class I, class II, and class III MHC proteins.
 The genes of the MHC exhibit genetic variability; and the MHC has several genes for each class
hence it is polygenic.
 The MHC is also polymorphic, meaning a large number of alleles exist in the population for each
of the genes.
 Therefore, a large number of alleles exist in the population for each of the genes. Each individual
inherits a restricted set of alleles from his or her parent. Sets of MHC genes tend to be inherited
as a block or haplotype. There are relatively infrequent cross-over events at this locus.
 The structure of the MHC class I have two domains that are distant from each other, made up of
two parallel α helices on top of a platform that is created by a β-pleated sheet. The general
structure looks like a cleft whose sides are formed by the α helices and the floor is β-sheet.
 Generally, the MHC molecules have a broad specificity for peptide antigens and many different
peptides can be presented by any given MHC allele binding a single peptide at a time.
 The α helices forming the binding clefts are the site of the amino acid residues that are
polymorphic (varying allelic forms) in MHC proteins, meaning that different alleles can bind and
present different peptide antigens. For all these reasons, MHC polymorphism has a major effect
on antigen recognition.
 The function of T-cells on interaction with the MHC molecules reveals that the peptide antigens
associated with class I MHC molecules are recognized by CD8+ cytotoxic T-lymphocytes (Tc cells)
and MHC class-II associated with peptide antigens that are recognized by CD4+ Helper T-cells (Th
cells).
 The MHC in humans is known as human leukocyte antigens (HLA) complex.

MICROBIOLOGY
HLA complex
In humans, the HLA complex of genes is located on short arm of chromosome 6 containing several genes
that are critical to immune function. The HLA complex of genes is classified into three classes as follows:
1. Class I: HLA-A, HLA-B, and HLA-C.
2. Class II: HLA-DR, HLA-DQ, and HLA-DP. All of these are present within HLA-D region of HLA
complex.
3. Class III: Complement loci that encode for C2, C4, and factor B of complement system and TNFs
alpha and beta.
Gene Products of HLA complex
Class I MHC genes encode glycoproteins expressed on the surface of nearly all nucleated cells; the major
function of the class I gene products is presentation of endogenous peptide antigens to CD8+ T cells.
Class II MHC genes encode glycoproteins expressed predominantly on APCs (macrophages, dendritic
cells, and B cells), where they primarily present exogenous antigenic peptides to CD4+ T cells.
Class III MHC genes encode several different proteins, some with immune functions, including
components of the complement system and molecules involved in inflammation.
Major Histocompatibility Complex (MHC) Types
In humans, the MHC molecules are divided into three types, Class I, Class II and Class III. Class I MHC
molecules are coded from three different locations called A, B and C and these molecules are expressed
in all nucleated cells. Class II MHC genes are located in the D region and there are several loci such as
DR, DQ and DP and these molecules are expressed only in antigen-presenting cells. Class III MHC genes
are coded in the region between Class I and Class II genes. Class III MHC genes codes for cytokines and
complement proteins which play an important role during the immune response.

MICROBIOLOGY
Class I MHC Molecule and Class II MHC Molecule
Class I MHC Molecule
 The structure of Class I MHC molecule consists of two polypeptide chains α and β. These two
chains are connected together by non-covalent bonds. The α chain is characterized as an
internal membrane glycoprotein with a molecular weight of 45000 Da (in humans). Β chain, on
the other hand, is an extracellular microglobulin with a molecular mass of 12kDa.
 The α chain is made up of approximately 350 amino acids and also divided into three globular
domains α1, α2 and α3. Each of these domains contains roughly 90 amino acids. The N terminal of
α chain is the place of α1 domain, while α2 and α3 are present after α1 The α2 domain is
characterized by the formation of a loop of 63 amino acids; the loop is formed due to intrachain
disulfide bond. α3 also contains a disulfide bond enclosing 86 amino acids. The α 1 and α2 domains
interact to form peptide-binding units of class I MHC molecule.
 Moreover, α chain also consists of a stretch of 26 hydrophobic amino acids which holds the α
chain on the plasma membrane. This transmembrane segment is present as a form of α helix at
the hydrophobic region of the plasma membrane. An intracellular domain or the carboxyl-
terminal of α chain is located inside the cell and it contains around 30-40 amino acids.
 In humans the β chain is non-polymorphic and it is dimorphic in mice. α 3 and β chain are
structurally similar to the immunoglobulin C domain and also characterized as a disulfide loop. A
peptide binding platform is formed by β plated sheets of α 1 and α2
 Tcyt Cell (cytotoxic T cell) has specificity towards cells containing peptides associated with Class I
MHC due to the presence of CD8 antigen on the surface of Tcyt Cell. CD8 antigen has an affinity
towards the α3 domain of Class I MHC molecules.
Class II MHC Molecule

MICROBIOLOGY
 Class II MHC molecules are heterodimers and characterized by two non-covalently connected
polypeptide chains. The chains are termed a heavy chain (α, 30kDa) and light chain (β, 26kDa).
 Similar to class I MHC molecules, class II MHC molecules are also characterized by an
extracellular amino-terminal domain, a transmembrane domain, and an intracellular carboxy-
terminal tail.
 The class II MHC molecules are expressed on the surface of the antigen-presenting cells such as
B cells, dendritic cells, and macrophages.
 The α chain is divided into two domains α 1 and α2, while the β chain is also divided into two
groups β1 and β2. The β2 domain is responsible for the binding of T cell co-receptor CD4. The
α1 and β1 domains, on the other hand, are involved in the formation of the antigen-binding sites.
Peptides containing 13-20 amino acids can bind at the antigen-binding site of class II MHC.
 The presence of disulfide bonds in α 2, β1, and β2 domains is also an important structural feature of
the class II MHC molecules.
Class III MHC Molecule
 There are several serum proteases which involve in the complement system that come under
the group of class III MHC molecules.
 Class III MHC molecules do not have any involvement in antigen presentation.
 The complement components such as asC2, C4A, and C4B, and factor B are the most important
compounds involved as class III MHC molecules. Apart from these tumor necrosis factors α and
β and some heat shock proteins also come under this category.
Distribution of MHC
Essentially, all nucleated cells carry classical class I molecules. These are abundantly expressed on
lymphoid cells, less so on the liver, lung, and kidney, and only sparsely on the brain and skeletal muscle.
In humans, the surface of the villous trophoblast lacks HLA-A and -B and bears HLA G, which does not
appear on any other body cell. Class II molecules are also restricted in their expression, being present
only on antigen-presenting cells (APCs) such as B-cells, dendritic cells, and macrophages and on the
thymic epithelium. When activated by agents such as interferon g, capillary endothelia, and many
epithelial cells in tissues other than the thymus, they can develop surface class II and increase
expression of class I.
They function as cell surface markers enabling infected cells to signal cytotoxic and helper T-cells.
Importance of MHC
1. Antibody molecules interact with antigen directly but the T-Cell Receptor (TCR) only recognizes
antigen presented by MHC molecules on another cell, the Antigen Presenting Cell. The TCR is
specific for the antigen, but the antigen must be presented on a self-MHC molecule.

MICROBIOLOGY
2. The TCR is also specific to the MHC molecule. If the antigen is presented by another allelic form
of the MHC molecule in vitro (usually in an experimental situation), there is no recognition by
the TCR. This phenomenon is known as MHC restriction.
Peptide antigens associated with class I MHC molecules are recognized by CD8 + cytotoxic T lymphocytes,
whereas class II-associated peptide antigens are recognized by CD4 + helper T cells.
Antigen Presentation and Processing
The T cells can recognize the foreign antigen when the antigen is attached to the MHC molecules as an
MHC peptide complex. The formation of the MHC-peptide complex requires the degradation of protein
antigen in several steps. The degradation process is known as antigen processing. These degraded
proteins are then attached to the MHC molecules inside the cell and then the MHC molecules are
transported to the membrane to present the antigen with the T cell.
Antigen Presentation Pathway: Class I MHC molecules (Cytosolic pathway)
 Class I MHC molecules involve in presenting intracellular or endogenous pathogens or antigens.

Intracellular pathogens refer to those organisms which live and replicates inside the host cell. An
example of this type of pathogen is a virus.
 Under normal condition the MHC class I molecules forms a complex with the self-peptides or
self-antigens. While, in case of any viral infection, the MHC class I molecules present the peptide
derived from the virus which is then further recognized by T cells.
 Cell components such as a nucleus, endoplasmic reticulum and Golgi apparatus play an
important role in antigen processing and presentation.
 When a virus infected a normal cell, the viral DNA moves inside the cell and produce viral
proteins with the help of the host cell mechanisms. The viral proteins are synthesized in the
cytosol.
 The cytoplasm also contains a cylindrical protein complex called the proteasome. The main
function of the proteosome is to degrade the unwanted or damaged protein into smaller
peptides. At the time of viral infection, the viral proteins interacted with the proteosomes
present in the cytoplasm. The processing took place in the cytosol and as a result, the proteins
are degraded into smaller peptides (8-15 amino acid long).
 In the next step, these fragmented peptides are transported into the endoplasmic reticulum.
The transport took place due to a peptide delivery system called the transporter associated with
antigen processing (TAP). TAP is made up of two domains or subunits called TAP 1 and TAP 2.
 Inside the endoplasmic reticulum the α and β chains of MHC class I molecules are synthesized
and by the help of a group of chaperone proteins, the MHC class I molecule is formed and
moves towards the TAP. As a result, the peptides bind at the peptide-binding site of the class I
MHC molecule inside the endoplasmic reticulum and forms the MHC class I-peptide complex.

MICROBIOLOGY
 In the next step, the MHC class I- peptide complex moves to the surface of the Golgi apparatus
and by the help of secretory vesicle, it moves towards the surface of the plasma membrane.
 Once the MHC class I-peptide complex reaches the cell surface, the T cell receptors recognize
the antigen peptide complex. Moreover, the co-receptor CD8 of the T cell attaches with the
α3 domain of the MHC class I molecule. Hence, the antigen is presented to the T cell.
Antigen Presentation Pathway: Class II MHC molecules (Endocytic Pathway)
 MHC class II molecules are responsible for presenting exogenous or extracellular pathogen or
antigen. The extracellular pathogen refers to the organisms which can grow and reproduce
outside of the host cell. Bacteria, exotoxins, parasites are examples of extracellular antigens.
These antigens are taken up by the cell by endocytosis or phagocytosis.
 Only the antigen-presenting cells involved in antigen processing and presentation by MHC class
II molecules. These cells include B cells, macrophages, and dendritic cells. The pathway took
place only after the engulfment of the antigen by the antigen-presenting cells.
 Inside the cell, the antigen carries a covering called an endosome. The endosome is fused with
the lysosome present in the cytoplasm and forms endolysosomes. As a result, the foreign
protein is degraded by the proteolytic enzyme present inside the lysosome and small peptides
are formed.
 The class II MHC molecules are synthesized and formed in the endoplasmic reticulum. The α and
β chain of the molecule is also associated with the invariant chain. This association helps to
restrict the binding of self-antigen with the class II MHC molecule. The invariant chain- MHC
complex is then transported from the endoplasmic reticulum to the Golgi apparatus and from
the Golgi apparatus to another vesicle. Inside the vesicle, the invariant chain is digested and only
a small fragment (Class II-associated invariant chain polypeptide: CLIP) is attached with the
molecule.
 In the next step, the vesicle containing the MHC class II molecule is then fused with the vesicle
containing fragmented peptides. The fragmented peptide is then bound with the MHC class II
molecule by displacing the CLIP. This newly formed MHC class II-peptide complex is then
transported to the surface of the cell.
 Once at the cell surface, the antigen is presented to the T cells. The T cell recognizes the peptide
bound with the MHC class II molecule by the help of the T cell receptor and the CD4 co-receptor
binds with the β2 domain of the class II MHC molecule.

MICROBIOLOGY

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KBCNMU conducted Science & Commerce College, Molgi

B.Sc. T.Y. MICROBIOLOGY

UNIT 1 HISTORY OF IMMUNOLOGY

The properties of antigens are as follows:

 The antigen should be a far off substance to induce an immune reaction .

 The antigens have a molecular mass of 14,000 – 6,00,000 Da.

 They are mainly proteins and polysaccharides.

 Antigens are species-specific.

On the Basis of Origin

There are different types of antigens on the idea of origin:

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Autoantigens are normal proteins or a posh of proteins or macromolecules that's attacked by a

A native antigen is the one that's not processed by an antigen-presenting cell.

On the Basis of Immune Response

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Difference Between Antigen and Antibody

Also called Immunogens Also called Immunoglobulins

Antigens are usually lipids. However, they

Protects against the effects of the

Antibodies always originate within the

What are Antibodies?

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An antibody structure consists of a Y-shaped molecule composed of 4 polypeptide subunits. The

What is an Antibody (Ab)?

– Also called immunoglobulins (Ig)

– Antigen binding site is called paratope.

What is an Antigen-Antibody Interaction?

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The antigen-antibody interaction is a bimolecular irreversible association between antigen and

Antigen-Antibody Interaction Properties

– Highly specific reaction

– Occurs in an observable manner

– No denaturation of antibodies and antigens

 The affinity of the antibody

 Valency(no. of binding sites) of antibody and antigen

 And the structural arrangement of epitopes and paratopes.

– Cross-Reactivity: It refers to the ability of an antibody to bind to similar epitopes of different

Stages of Antigen-Antibody Interaction

1. Primary Stage: It is the initial interaction between antigens and antibodies.

– Without any visible effects

2. Secondary Stage: It is the irreversible interaction between antigens and antibodies.

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– With visible effects

Factors Affecting Antigen-Antibody Interaction

5. Concentrations of Ag and Ab: Increase in the concentration of antigen and antibody enhances

Chemical Bonds Responsible for the Antigen-Antibody Reaction

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Strength of Ag-Ab interactions

 The combined strength of total non-covalent interactions between a single Ag-binding site of Ab

 Low-affinity Ab: Bind Ag weakly and dissociates readily.

 High-affinity Ab: Bind Ag tightly and remain bound longer.

 Antibodies elicited by one Ag can cross-react with unrelated Ag if they share identical epitopes

Types of Antigen-Antibody Interaction

Ag-Ab reactions are basically of two types:

1. In Vivo (Occurring in natural condition): It includes the general antibody-mediated immune

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 Ab Dependent Cell-Mediated Toxicity

– In the presence of electrolytes

– At specific pH and Temperature

– Antibodies involved are called precipitins

– Types: In solution (Flocculation and Ring Test)

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