Unit 3

Types of immunity- Humoral immunity
and Cellular immunity
Dr. LAKSHMI
ASSISTANT PROFESSOR
KIET School Of Pharmacy,KIET,
GHAZIABAD

Immunity
• Immunity generally means protection.
• In biological terminology, immunity is the natural process that
is responsible for fighting microorganisms, which enter our
bodies to damage the cells.
• Basically, when our body detects a pathogen, our immune
system gets activated.
• Pathogens are the microorganisms that are either bacteria or
viruses, which are capable of causing a disease.

Immunity
• The Immune System has 3 Lines of Defense Against Foreign Pathogens:
• 1. Physical and Chemical Barriers (Innate Immunity)
• 2. Nonspecific Resistance (Innate Immunity)
• 3. Specific Resistance (Acquired Immunity)

Mechanism of Innate Immunity
• Epithelial surfaces
• Antibacterial substances
• Cellular factors
• Inflammation
• Fever
• Acute phase proteins

• Epithelial surfaces: Skin
• Provides mechanical barrier to microorganisms
• Provides bactericidal secretions
• The resident bacterial flora of skin and mucous surfaces prevent
colonization by pathogen
• Alteration of normal flora may lead to invasion by extraneous
microbes and cause serious diseases. e.g, clostridial enterocolitis
following oral antibiotics.

• Respiratory tract:
• Respiratory tract is lined by moist mucous surfaces which act as
trapping mechanism.
• Inhaled particles are arrested in nasal passage on moist mucous
membrane surfaces.
• The hair like cilia propels the particles towards pharynx and are
swallowed or coughed out.
• Some particles which manage to reach alveoli are ingested by
phagocytes

• Intestinal tract:
• Saliva present in mouth inhibits many microorganisms.
• Acidic pH of gastric juices destroys the swallowed bacteria
• Normal flora of intestine prevent colonization of pathogens.
• Conjunctiva:
• Tears flush away bacteria and other dust particles
• lysozyme present in tears has bactericidal action.

• Genitourinary tract:
• Urine eliminate bacteria from urethra by its flushing action.
• Acidic pH of vaginal secretion of female due to fermentation
of glycogen by lactobacilus makes vagina free from
microorganisms.
• In males, semen is believed to have some antibacterial
substance.

• Antibacterial substances in blood and tissues:
• Beta lysin: relatively thermostable substance active against
anthrax and related bacilli.
• Basic Polypeptide: e.g., leukins and plakins
• Acidic substances: lactic acid present in tissue and infected
area
• Interferon : protects against certain acute and viral
infections.

• Cellular factors:
• Once the infective agent cross the epithelial barriers, tissue factors come
into play for defense.
• Process:
• Invasion of tissues by infective agent
• Accumulation of phagocytes in site of infection
• Deposition of fibrin that entangles the organisms (act as barrier to
spread of infection)
• Phagocytic cells ingest these organisms and destroy them.

• Inflammation:
• An important non-specific defense mechanism
• Occurs as a result of tissue injury, initiated by entry of pathogens.
• Leads to vasodilation, increased vascular permeability and cellular
infiltration
• Due to increased vascular permeability, plasma pours out and dilutes
the toxic products present.
• Fibrin barrier is laid to wall off the site of infection

• 5.Fever:
• Rise in temperature following infection is natural defense mechanism.
• Destroys the infecting organism
• Stimulates the production of interferon, which help in recovery from
viral infections .

• 6. Acute phase proteins:
• After injury ,there is sudden increase or decrease in plasma
concentration of certain proteins, collectively called Acute phase
proteins
• E.g. C reactive protein (CRP),Mannose binding proteins etc.
• They activate the alternative pathway of complement
• Prevent tissue injury and promote repair of inflammatory lesions

Adaptive or Acquired immunity
• The adaptive immune system, also called acquired immunity, uses
specific antigens to strategically mount an immune response.
• Unlike the innate immune system, which attacks only based on the
identification of general threats, the adaptive immunity is activated by
exposure to pathogens, and uses an immunological memory to learn
about the threat and enhance the immune response accordingly.
• The adaptive immune response is much slower to respond to threats and
infections than the innate immune response, which is primed and ready
to fight at all times.

Adaptive or Acquired immunity
• Antigen specificity: immune system or antibodies can distinguish among
antigens, even between two proteins that differ in only one amino acid.
• Diversity: immune system is capable of generating large antibody
diversity in its recognition molecules.
• Immunologic memory: immune system exhibits memory on second
encounter of same antigen by generating a secondary response which is
more specific m quick.
• Self/non-self recognition: does not react with body’s own molecule but
effectively eliminates foreign antigens.

Cells of the adaptive immune system
• Unlike the innate immune system, the adaptive immune system relies on
fewer types of cells to carry out its tasks: B cells and T cells.
• Both B cells and T cells are lymphocytes that are derived from specific
types of stem cells, called multipotent hematopoietic stem cells, in the
bone marrow.
• After they are made in the bone marrow, they need to mature and
become activated.
• Each type of cell follows different paths to their final, mature forms.

• B cells
• After formation and maturation in the bone marrow (hence the name “B cell”),
the naive B cells move into the lymphatic system to circulate throughout the
body.
• In the lymphatic system, naive B cells encounter an antigen, which starts the
maturation process for the B cell.
• B cells each have one of millions of distinctive surface antigen-specific receptors
that are inherent to the organism’s DNA.
• For example, naive B cells express antibodies on their cell surface, which can also
be called membrane-bound antibodies.

• When a naive B cell encounters an antigen that fits or matches its membrane-
bound antibody,
• It quickly divides in order to become either a memory B cell or an effector B cell,
which is also called a plasma cell.
• Antibodies can bind to antigens directly.
• The antigen must effectively bind with a naive B cell’s membrane-bound antibody
in order to set off differentiation, or the process of becoming one of the new
forms of a B cell.

• Memory B cells express the same membrane-bound antibody as the
original naive B cell, or the “parent B cell”.
• Plasma B cells produce the same antibody as the parent B cell, but they
aren’t membrane bound.
• Instead, plasma B cells can secrete antibodies.
• Secreted antibodies work to identify free pathogens that are circulating
throughout the body.
• When the naive B cell divides and differentiates, both plasma cells and
memory B cells are made.

• B cells also express a specialized receptor, called the B cell receptor (BCR).
• B cell receptors assist with antigen binding, as well as internalization and
processing of the antigen.
• B cell receptors also play an important role in signaling pathways.
• After the antigen is internalized and processed, the B cell can initiate signaling
pathways, such as cytokine release, to communicate with other cells of the
immune system.

• T cells
• Once formed in the bone marrow, T progenitor cells migrate to the thymus
(hence the name “T cell”) to mature and become T cells.
• While in the thymus, the developing T cells start to express T cell receptors
(TCRs) and other receptors called CD4 and CD8 receptors.
• All T cells express T cell receptors, and either CD4 or CD8, not both. So, some T
cells will express CD4, and others will express CD8.

• Unlike antibodies, which can bind to antigens directly, T cell receptors can only
recognize antigens that are bound to certain receptor molecules, called Major
Histocompatibility Complex class 1 (MHCI) and class 2 (MHCII).
• These MHC molecules are membrane-bound surface receptors on antigen-
presenting cells, like dendritic cells and macrophages.
• CD4 and CD8 play a role in T cell recognition and activation by binding to either
MHCI or MHCII.

• There are three types of mature T cells:
• Helper T cells express CD4, and help with the activation of TC B cells, and other
immune cells
• Cytotoxic T cells express CD8, and are responsible for removing pathogens and
infected host cells.
• T regulatory cells express CD4 and another receptor, called CD25. T regulatory
cells help distinguish between self and nonself molecules, and by doing so,
reduce the risk of autoimmune diseases.

Regulatory, help
distinguish between
self and nonself
molecules
Memory

Humoral vs. Cell Mediated Immunity
• Immunity refers to the ability of your immune system to defend against infection and
disease. There are two types of immunity that the adaptive immune system provides,
and they are dependent on the functions of B and T cells, as described above.
• Humoral immunity is immunity from serum antibodies produced by plasma cells.
• More specifically, someone who has never been exposed to a specific disease can gain
humoral immunity through administration of antibodies from someone who has been
exposed, and survived the same disease. “Humoral” refers to the bodily fluids where
these free-floating serum antibodies bind to antigens and assist with elimination.

Humoral vs. Cell Mediated Immunity
• Cell-mediated immunity can be acquired through T cells from someone who is immune
to the target disease or infection.
• “Cell-mediated” refers to the fact that the response is carried out by cytotoxic cells.
Much like humoral immunity, someone who has not been exposed to a specific disease
can gain cell-mediated immunity through the administration of TH and TC cells from
someone that has been exposed, and survived the same disease.
• The TH cells act to activate other immune cells, while the TC cells assist with the
elimination of pathogens and infected host cells.

Immunological memory
• Because the adaptive immune system can learn and remember specific
pathogens, it can provide long-lasting defense and protection against recurrent
infections.
• When the adaptive immune system is exposed to a new threat, the specifics of
the antigen are memorized so we are prevented from getting the disease again.
• The concept of immune memory is due to the body’s ability to make antibodies
against different pathogens.

Immunological memory
• A good example of immunological memory is shown in vaccinations.
• A vaccination against a virus can be made using either active, but
weakened or attenuated virus, or using specific parts of the virus that
are not active. Both attenuated whole virus and virus particles cannot
actually cause an active infection.
• Instead, they mimic the presence of an active virus in order to cause
an immune response, even though there are no real threats present.
• By getting a vaccination, you are exposing your body to the antigen
required to produce antibodies specific to that virus, and acquire a
memory of the virus, without experiencing illness.

Innate Immunity vs. Adaptive Immunity
Attribute Innate Immunity Adaptive Immunity
Response Time Fast: minutes or hours Slow: days
Specificity
Only specific for molecules and molecular
patterns associated with general pathogens
or foreign particles
Highly specific! Can discriminate between
pathogen vs. non-pathogen structures, and
miniscule differences in molecular structures
Major Cell Types
Macrophages, Neutrophils, Natural Killer
Cells, Dendritic Cells, Basophils, Eosinophils
T cells, B cells, and other antigen presenting
cells
Key Components
Antimicrobial peptides and proteins, such as
toxic granules
Antibodies
Self vs. Nonself Discrimination
Innate immunity is based on self vs. nonself
discrimination, so it has to be perfect
Not as good as the innate immune system,
but still pretty good at determining which is
which.
Problems in self vs. nonself discrimination
result in autoimmune diseases
Immunological Memory None
Memory used can lead to faster response to
recurrent or subsequent infections
Diversity and Customization
Limited: Receptors used are standard and
only recognize antigen patterns. No new
receptors are made to adapt the immune
response
Highly diverse: can be customized by genetic
recombination to recognize epitopes and
antigenic determinants.

Humoral immune response
• Many of the bacteria that cause infectious disease in humans multiply in the
extracellular spaces of the body, and most intracellular pathogens spread by
moving from cell to cell through the extracellular fluids.
• The extracellular spaces are protected by the humoral immune response, in
which antibodies produced by B cells cause the destruction of extracellular
microorganisms and prevent the spread of intracellular infections.
• The activation of B cells and their differentiation into antibody-secreting
plasma cells is triggered by antigen and usually requires helper T cells.

Antibody functions
• Antibodies contribute to immunity in three main ways
• Neutralization
• To enter cells, viruses and intracellular bacteria bind to specific
molecules on the target cell surface. Antibodies that bind to the
pathogen can prevent this and are said to neutralize the pathogen.
• Neutralization by antibodies is also important in preventing bacterial
toxins from entering cells.

Antibody functions
• Opsonization.
• Antibodies protect against bacteria that multiply outside cells mainly by
facilitating uptake of the pathogen by phagocytic cells that are specialized to
destroy ingested bacteria.
• Antibodies do this in either of two ways. In the first, bound antibodies coating
the pathogen are recognized by Fc receptors on phagocytic cells that bind to
the antibody constant C region.
• Coating the surface of a pathogen to enhance phagocytosis is called
opsonization.

Antibody functions
• Complement activation
• Alternatively, antibodies binding to the surface of a pathogen can activate the proteins
of the complement system.
• Complement activation results in complement proteins being bound to the pathogen
surface, and these opsonize the pathogen by binding complement receptors on
phagocytes.
• Other complement components recruit phagocytic cells to the site of infection, and
the terminal components of complement can lyse certain microorganisms directly by
forming pores in their membranes. Which effector mechanisms are engaged in a
particular response is determined by the isotype or class of the antibodies produced.

Cell mediated immunity
• Unlike B cells, T lymphocytes (T cells) are unable to recognize pathogens without
assistance.
• First, an antigen-presenting cell (APC, such as a dendritic cell or a macrophage )
detects, engulfs (via phagocytosis in the case of macrophages or by entry of the
pathogen of its own accord in the case of dendritic cells), and digests pathogens into
hundreds or thousands of antigen fragments.
• These fragments are then transported to the surface of the APC, where they are
presented on proteins known as Major Histocompatibility Complexes class II (MHC II)

• T cells become activated towards a certain antigen once they encounter
it displayed on an MHC II.
• After a virus or bacteria enters a cell, it can no longer be detected by the
humoral immune response. Instead, the cellular immune response must
take over.
• To do so, a T cell will become activated by interacting with an antigen of
the infecting cell or virus presented on the MHC II of an APC.

• Cytotoxic T cells
• TC cells attempt to identify and destroy infected cells by triggering
apoptosis (programmed cell death) before the pathogen can replicate
and escape, thereby halting the progression of intracellular infections.
• To recognize which cells to pursue, TC recognize antigens presented on
MHC I complexes, which are present on all nucleated cells.
• TC cells also support NK lymphocytes to destroy early cancers.

• Helper T cells
• Cytokines are signaling molecules secreted by a TH cell in response to a
pathogen-infected cell; they stimulate natural killer cells and phagocytes
such as macrophages.
• Phagocytes will then engulf infected cells and destroy them.
• Cytokines are also involved in stimulating TC cells, enhancing their ability
to identify and destroy infected cells and tumors.

Structure of Immunoglobulins
Dr. LAKSHMI
ASSISTANT PROFESSOR
GHAZIABAD

Immunoglobulins
• Immunoglobulins (Ig) or antibodies are glycoproteins that are produced by
plasma cells.
• B cells are instructed by specific immunogens, for, example, bacterial proteins, to
differentiate into plasma cells, which are protein-making cells that participate in
humoral immune responses against bacteria, viruses, fungi, parasites, cellular
antigens, chemicals, and synthetic substances.
• Immunoglobulins constitute about 20% of the protein in plasma.

Basic immunoglobulin Function
• All antibodies exhibit one or more functions
(bifunctional) including activation of the complement
system, opsonization of microbes to be easily
phagocytosed, prevention of attachment of the
microbes to mucosal surfaces, and neutralization of
toxins and viruses

Immunoglobulins
• All immunoglobulin molecules basically consist of :
• two identical heavy chains and
• two identical light chains
• held together by disulfide linkages (Inter-chain and Intra-chain) and non-covalent
interactions.
• It is Y shaped tetramer (H2L2)
• Each heavy chain contains 450 amino acids Light chain has 212 amino acids
• Heavy chain of Ig are linked to carbohydrates hence Ig are glycoproteins.

Variable and constant regions
• Each chain of Ig has two regions – constant and variable region.
• Light chain
• Amino terminal half is variable region (VL )
• carboxy terminal half is constant region (CL)
• Heavy chain
• One quarter of amino terminal region – variable region (VH)
• Remaining three quarters – constant region (CH1 , CH2 , CH3 )

Immunoglobulins classes
• Two types of light chains : kappa () and lambda (λ)
• An Ig contains two () or two λ light chain and never a mixture.
• Kappa chain (60%) is more common in human.
• IgG
• IgM
• IgA
• IgD
• IgE
- Gamma heavy chains
- Mu heavy chains
- Alpha heavy chains
- Delta heavy chains
- Epsilon heavy chains

IgG Immunoglobulins
• Major serum Ig – 75 -80 %
• Single Y shaped unit (monomer)
• It is the antibody seen in secondary immuno response.
• It can transverse blood vessels readily
• IgG is only Ig that can cross the placenta and transfer the mothers immunity to
developing fetus.

IgM Immunoglobulins
• Largest Ig composed of 5 Y shaped units held together by a J
polypeptide chain.
• Pentamer –bind with 5 antigenic sites
• Due to its large size, IgM cannot transverse blood vesssels, hence it
is restricted to the blood stream.
• IgM is first antibody to be produced in response to an antigen and is
most effective against invading micro-organism.

IgM Immunoglobulins
• Ig M are predominant class of Ab produced in primary response to
an Ag.
• Natural Ab are IgM in nature.
• A person having blood group A Antigen will have anti B antibodies in
his circulation (isohemagglutinins). These are produced without any
known antigenic stimulation and hence called natural antibodies.
• IgM Ab cannot cross placenta
• So if the fetus even though it carries an incompatible Ag , is
protected from natural Ab of the mother.

IgA Immunoglobulins
• Single (monomer) or double unit (dimer) held together by J chains
• Mostly found in body secretions such as saliva, tears, sweat, milk
and the walls of intestine.
• Most predominant Ab in colostrum.
• Ig A molecules bind with bacterial Ag present on body surface and
remove them. So IgA prevents the foreign substances from entering
the body cells.

IgA Immunoglobulins
• The dimer are stabilized against proteolytic enzymes by secretory piece.
• The secretory piece is produced in liver, reaches to the intestinal mucosal cells,
where it combines with Ig A dimer to form the Secretory IgA which is then
released.
J Chain
Secretory Piece

IgE Immunoglobulins
• Single Y shaped (Monomer)
• Normally present in minute conc in blood – 0.3g/ml
• IgE levels are elevated in individuals with allergies as it is associated with the
body’s allergic response – Hay fever, Asthma, Anaphylactic shock.
• IgE tightly binds with Fc receptors on basophils and mast cells which release
histamine and cause allergy.
• Immediate type Hypersensitivity reaction – peak at 30 min

IgD Immunoglobulins
• Single Y shaped unit (Monomer)
• Present in low concentration in circulation. Present on surface of B cells
• Their function is not known

Functions of Immunoglobulins
Immunoglobulin Major Functions
IgG
 Main antibody in the secondary response.
 Opsonizes bacteria, making them easier to phagocytose.
 Fixes complement, which enhances bacterial killing.
Neutralizes bacterial toxins and viruses.
 Crosses the placenta.
IgA  Secretory IgA prevents attachment of bacteria and viruses to
mucous membranes.
 Does not fix complement.

Functions of Immunoglobulins
IgM  Produced in the primary responseto an antigen.
 Fixes complement.
 Does not cross the placenta.
IgD  Found on the surfaces of B cells where it acts as a receptor
for antigen.
IgE
 Mediates immediate hypersensitivity (allergy) by causing
release of mediators from mast cells and basophils upon
exposure to antigen (allergen).
 Does not fix complement.
 Main host defense against helminthic infections.

Structure and function of MHC
Dr. LAKSHMI
ASSISTANT PROFESSOR
KIET School Of Pharmacy, KIET,
GHAZIABAD

Introduction
• Major Histocompactibility complex (MHC) is set of surface proteins located on
the cell membrane of nucleated cells.
• It plays more important work to identify the antigen between self and non
self body, intracellular recognization and responsible for antigen
presentation.
• Histo refers to tissues. Compatibility refers to living together harmoniously.
• MHC molecules always recognize only T lymphocytes. The two types of MHC
are worked in immunity. T helper (Th) cell recognized by MHC molecules II,
and T cytotoxic (Tc) cells are recognized by MHC I molecules.

Introduction
• Definition
• “Major Histocompatibility complex is membrane attached protein
which work on recognization of antigen between self and non self body
and antigen presentation”.
• Peter Gorer (1930) found that four group of MHC molecules he used the
blood sample of mice to identified the blood group antigen which
designated by I to IV group of MHC.
• Georg Snell, Jean Dausset and Bariy received noble prize in 1980 for
their contribution to the discovery of MHC molecule.

Classes of MHC molecules
• The MHC molecules are classified in to four classes namely ;-
• Class I MHC molecules: found on all nucleated cells (not RBCs)
• Class II MHC molecules: found on APC, Dendritic cells, Macrophages, B cells, other
cells
• Class III MHC molecules
• Class IV MHC molecules
• T cell receptors recognize antigenic peptide/MHC complexes
• CD4+ T cells: restricted by class II
• CD8+ T cells: restricted by class I

Class I MHC molecules
• Class I MHC(45 KD) molecule are a group of major histocompactibility
antigen.
• They are present on the surface of all nucleated cells except nervous
tissue and platelets.
• It present antigen to Tc cells.
• It bind with CD-8 adhesion molecules of Tc cells.
• It brings about cell mediated immune response.

Structure of Class I MHC molecules
• It consists two polypeptide chains namely α chain and β2 – micro globulin.
• α chain which is non covalently attached with β2 microglobuline .
• α chain contain a transmembrane glycoprotein which is encoded by A,B and C gene of
grouped HLA.
• α chain is organized by three domains such as α 1, α 2 and α 3 each domain containing
90 amino acids sequences .
• β2 microglobuline is similar in size of α 3 and it dose not contain trans membrane
proteins .
• When the antigen is internalized and processed inside by proteosome (Ubiquitin,
cytosolic degradation), the peptides are produced .

Class II MHC molecules
• Class II MHC molecule are present on the surface of antigen
presenting cell and cell which engulfed the foreign antigen.
• It binds with the exogenous(endocytic degradation ) antigens.
• It binds with CD4 adhesion molecules TH cells.
• It also consist of two polypeptide chains namely α chain and β chain.
• Antigen is processed inside the endosome and peptide is further
loaded on groove of MHC II molecules.

Structure of Class II MHC molecules
• The class II MHC Molecule consists of two polypeptide chain namely
α chain (33 kDa) and β (28kDa) chain.
• The both chain are attached noncovalently.
• Each chain contains two units. The two units of α chain are called
α1 and α2. The two domains of β chains are called β1 and β2.
• β2 and α2 are transmembrane domains anchoring the MHC to
plasma membrane.
• The α1 and β1 domains jointly bear a peptide binding groove

Class IIIMHC molecules
• The molecules include complements like C2 and C4 and Bf (factor
B).
Class IV MHC molecule
• These molecule is present on T cells of leukemia(Tla) as well as
on immature thymocytes .

Antigen Processing
• T-independent antigen
• Large antigen molecules with readily accessible, repeating antigenic
determinants
• B cells can bind these directly without being processed
• Stimulates B cells to differentiate into a plasma cell and produce antibodies

Antigen Processing
• T-dependent antigen
• Smaller antigens with less accessible antigenic determinants
• B cells require involvement from helper T cells to target these antigens
• Helper T cells are assisted by leukocytes that process the antigen to
make the antigenic determinants more accessible
• Processing is different based on whether the antigen is exogenous or
endogenous

Processing of exogenous antigen
• APC internalizes the invading pathogen and enzymatically digests it
into smaller antigenic fragments which are contained within a
phagolysosome
• Phagolysosome fuses with a vesicle containing MHCII molecules
• Each fragment binds to the antigen-binding groove of a
complementary MHCII molecule
• The fused vesicle then inserts the MHCII- antigen complex into the
cytoplasmic membrane so the antigen is presented on the outside the
cell

Processing of endogenous antigen
• The intracellular pathogens are also digested into smaller
antigenic determinants
• Each fragment binds to a MHCI molecule located in the
endoplasmic reticulum membrane
• The membrane is packaged into a vesicle by a Golgi body which is
inserted into the cytoplasmic membrane so the antigen is
displayed on the cell’s surface

HLA -human leukocyte antigen.
• HLA is the human leukocyte antigen.
• HLA is the MHC molecules present in human beings.
• HLA is a set of surface protein present on the surface of all nucleated cells.
• They are responsible for graft rejection, adaptive immunity, defense against
infection, some time it is expressed on cancer cell destruction, certain
autoimmune diseases and certain complements.
• MHC is the general term referring to the cell surface antigen of vertebrates.

Functions of MHC
• MHC molecules are loaded with a bit of sample peptide fragment
derived from the degradation of proteins present inside the cell. This
peptide is the mirror image of proteins present inside the cell.
• MHC molecules contain self as well as nonself (foreign) antigen.
• They bring about defense against infections and diseases.
• They mediate certain autoimmune diseases.
• They are responsible for individual smell of people.

MHC Class I MHC Class II
Structure
MHC class I molecules consist of
one membrane-spanning α chain
(heavy chain) produced by MHC
genes, and one β chain (light chain
or β2-microglobulin) produced by
the β2-microglobulin gene.
MHC class II molecules consist of
two membrane-spanning chains, α
and β, of similar size and both
produced by MHC genes.
Types of APCs
MHC I glycoproteins are present in
all nucleated cells.
MHC II glycoproteins are only
present on specialised antigen-
presenting cells (APCs), including
macrophages that engulf foreign
particles such as bacteria, dendritic
cells that present antigen to T cells,
and B cells that produce
antibodies.
Nature of Antigen Presentation
MHC class I glycoproteins
present endogenous antigens that
originate from the cytoplasm.
MHC II proteins present
exogenous antigens that originate
extracellularly from foreign bodies
such as bacteria.
Size of peptide
MHC Class I present 8-10 amino
acid peptides
MHC Class II presents 14-18 amino
acid peptides.
Comparative overview

MHC Class I MHC Class II
Responsive T Cells
Present antigen to cytotoxic T cell
lymphocytes (CD8+ T Cells);
Present antigen to helper T cell
lymphocytes; (CD4+ T cells).
Co-receptor responsible
Binds with CD8 coreceptors
molecules on cytotoxic T cells
Binds with CD4 co-receptors
molecules on helper T cells
Sources of Protein Antigens
Cytosolic proteins (mostly
synthesized in the cell, may enter
cytosol from phagosomes)
Endosomal/lysosomal proteins
(mostly internalized from
extracellular environment)
Enzymes Responsible for peptide
generation
Cytosolic proteasome
Endosomal and lysosomal
proteases (e.g., cathepsins)
Site of peptide loading of MHC Endoplasmic reticulum Specialized vesicular compartment
Molecules involved in transport of
peptides and loading of MHC
molecules
Chaperones, TAP in ER
Chaperones in ER; invariant chain
in ER, Golgi and MHC Class II
compartment/Class II vesicle; DM
End Result
Presentation of foreign-
intracellular antigens or altered
self-antigens; targets cell for
destruction
Presentation of foreign
extracellular antigens; induces
antibody production, and attracts
immune cells to area of infection
Comparative overview

Summary
• The both MHC I and II molecule are responsible for antigen presentation and it
has application of antigen recognization between self and nonself recognization,
mostly they are located on T lymphocytes encoded by chromosome 6 of the
human. The two types of antigen degraded peptides (exogenous and
endogenous) are involved to complete these process of antigen neutralization.

Hypersensitivity reactions
Dr. LAKSHMI
ASSISTANT PROFESSOR
KIET School Of Pharmacy, KIET,
GHAZIABAD

• Hypersensitivity reactions are an overreaction of the immune system to
an antigen which would not normally trigger an immune response.
• The vulnerability of an individual to these reactions can have a genetic
link. Overreaction to innocuous antigens are linked to changes in the CD
regions of T-helper cell membranes, explaining why reactions like peanut
allergies can commonly run in families.
• Overreaction to self-antigens is normally due to a failure in central
tolerance, and this failure can also have genetically-inheritable features.

• As is the case for many immune reactions, hypersensitivity reactions require
two separate interactions of the immune system with the antigen.
• The first time an antigen enters the body, it is picked up by antigen-presenting
cells (such as macrophages or dendritic cells) and taken to the nearest lymph
node, where it is presented to naïve T-cells.
• Cross-linking of the antigen with T-cells, as well as co-stimulatory molecules, can
lead to activation of that T-cell and subsequent differentiation into “primed”
Th1, Th2, or Th17 cells, which are specific to that antigen
• can stimulate further immune responses if they meet the antigen again. It is
this second meeting that could result in a hypersensitivity reaction.

Hypersensitivity reactions: Types
• According to the Coombs and Gell classification, there are four main types of
hypersensitivity reaction.
• Type I
• Type II
• Type III
• Type IV

Type I Hypersensitivity reactions
• First interaction with antigen
• Mast-cell activation is induced by secretion of IgE antibodies.
• Initial exposure to the antigen causes the priming of Th2 cells,
• their release of IL-4 causes the B cells to switch their production of
IgM to IgE antibodies which are antigen-specific.
• The IgE antibodies bind to mast cells and basophils, sensitising them
to the antigen.

• Antigen enters the body again,
• it cross links the IgE bound to the sensitised cells,
• causing the release of preformed mediators including histamine,
leukotrienes and prostaglandins.
• This leads to widespread vasodilation, bronchoconstriction, and increased
permeability of vascular endothelium.

• The reaction can be divided into two stages
• immediate, in which release of pre-
formed mediators causes the immune
response, and
• late-phase response 8-12 hours later,
where cytokines released in the
immediate stage activate basophils,
eosinophils, and neutrophils even
though the antigen is no longer present.

• Clinical Relevance - Anaphylaxis
• Anaphylaxis is a systemic response to an antigen, leading to
bronchoconstriction and vasodilation.
• This decline in oxygen transportation and can lead to anaphylactic
shock and possibly death.
• It is usually treated with adrenaline, to dilate the bronchioles and
constrict the blood vessels, antihistamines, to reduce the
inflammatory effects of histamine, and corticosteroids, to reduce
systemic inflammation.

Type II Hypersensitivity reactions
• Mediated by antibodies which target antigens on cell surfaces.
• When cell surface antigens are presented to T cells, an immune
response is started, targeting the cells to which the antigens are
attached.
• Antibodies binding to cells can activate the complement system, leading
to degranulation of neutrophils, a release of oxygen radicals, and
eventual formation of membrane attack complex – all of which lead to
destruction of the cell.
• Parts of the complement activation can also opsonise the target cell,
marking it for phagocytosis.

• The destruction of host cells in this way can lead to tissue-specific
damage.
• Type 2 hypersensitivity reactions may occur in response to host cells
(i.e. autoimmune) or to non-self cells, as occurs in blood transfusion
reactions.
• Type 2 is distinguished from Type 3 by the location of the antigens – in
Type 2, the antigens are cell bound, whereas in Type 3 the antigens
are soluble.

• Clinical Relevance - Acute Transfusion
Reactions
• Acute transfusion reactions are when an
inappropriate blood transfusion is
administered and a patient is given
blood not matching their ABO type.
• This leads to activation of complement
and widespread haemolysis by tumour
necrosis factor and other interleukins,
which can be fatal..

Type III Hypersensitivity reactions
• Type 3 hypersensitivity reactions are mediated by antigen-antibody
complexes (formed by soluble antigens) in the circulation that may be
desposited in and damage tissues.
• The complexes may become lodged in the basement membranes of
tissues which have particularly high rates of blood filtration – the kidney
and synovial joints being common targets.
• Once lodged, the immune complexes rapidly and significantly activate
the complement chain, causing local inflammation and attraction of
leucocytes.
• Activation of complement results in increased vasopermeability, the
attraction and degranulation of neutrophils, and the release of oxygen
free radicals which can severely damage surrounding cells.

Type III Hypersensitivity reactions
• Clinical Relevance - Rheumatoid Arthritis
• Rheumatoid arthritis can occur when antigen-antibody complexes
circulate in the bloodstream end up lodging in the complex filtration
systems responsible for maintaining the levels of synovial fluids at
synovial joints.
• The lodged immune complexes can cause a local inflammatory
response, leading to stiffness and pain in affected joints.

Type IV Hypersensitivity reactions
• Type 4 hypersensitivity reactions are mediated by antigen-specific
activated T-cells. When the antigen enters the body, it is processed by
antigen-presenting cells and presented together with the MHC II to a Th1
cell.
• If the T-helper cell has already been primed to that specific antigen, it will
become activated and release chemokines to recruit macrophages and
cytokines such as interferon-γ to activate them.

• Activated macrophages release pro-inflammatory factors, leading to local
swelling, oedema, warmth, and redness.
• They also secrete lysosomal elements and reactive oxygen species, again leading
to local tissue damage.
• CD8+ T cells may be involved in type 4 reactions where a foreign antigen is
detected on a cell, such as in organ rejection: this is known as cell mediated
cytotoxicity, and also results in recruitment and activation of macrophages.
• This reaction is also known as delayed-type hypersensitivity due to its
characteristic longer time period to appear following antigen exposure. The
reaction takes longer than all other types because of the length of time required
to recruit cells to the site of exposure – around 24 to 72 hours.

• Clinical Relevance - Contact Dermatitis
• Contact dermatitis can result from a wide variety of innocuous
substances, such as nickel, poison ivy, or household cleaning
products.
• Because of the delay in transporting Th1 cells to the site of
infiltration, symptoms can develop several days after initial exposure
to the substance, but redness, itching, swelling, and heat are all
common.

Immune stimulation and Immune suppression

Immune stimulation
• Immunostimulants are the substances that increases the ability of the immune
system to fight against infection & disease.
• Many compounds (drugs , vitamins &immune system components) can
stimulate our immune system against different micro-organisms.
• These drugs are useful in infection, immunodeficiency & cancer.

Immune stimulation
• Based on action there are two types of immunostimulants:
• Specific Immuno stimulants: Provide antigenic specificity in immune response
such as vaccination.
• Eg: Vaccine.
• Non-specific Immuno stimulants: These act irrespective of antigen specificity , so
act against all antigens.
• Eg: Immunoglobulins.

Types of immunstimulants
• Immunostimulants activate different elements of the immune system in
humans and animals.
• They develop the non-specific immunotherapy and immuno prevention by
stimulating the major factors of the immune system including phagocytosis,
properdin and complement systems, protective secretory IgA antibodies, α-
and γ-interferon release, T- and B-lymphocytes, synthesis of specific antibodies
and cytokines, and synthesis of pulmonary surfactant.

Types of immunstimulants
• There are several reasons for using the immunostimulants in the control of
various infectious diseases including:
• antibiotic resistance of the bacteria;
• allergic reactions to antibiotics;
• immunosuppressive effects of antibiotics; and
• Poor effects of the antibiotics in viral infections

Classification of immunostimulants
• Vaccines - Poliomyelitis vaccine , Rota virus vaccine.
• Adjuvants.
• Immunoglobulins.
• Miscellaneous agents used as stimulants:
• Levamisole.
• Thalidomide.
• Isoprinosine.
• Immunocynin,.

Vaccines
• Vaccine is a biological preparation that improves immunity to a particular disease.
Vaccine contain certain agents which stimulates immune system to recognize the foreign
agents.
• Ex: BCG vaccine for tuberculosis.
• Vaccines are suspensions of dead microorganisms (inactivated) or attenuated but live.
• In general live, attenuated vaccines are antigenically more potent than the inactivated
vaccines.
• Live vaccines achieve lifelong immunity with single dose but multiple doses are
required with polio vaccine.

Vaccines
• ROTA VIRUS VACCINE:
• It is a vaccine used to protect against rotavirus infections.
• These viruses are the leading cause of severe diarrhea among young children (3 years).
• The available rotavirus vaccine is human bovine reassortant vaccine.
• It is safe and effective. The oral vaccine is administered in three, monthly doses from
the age of 6-12 weeks to the age of 32 weeks.
• It may be with other vaccines.
• Adverse Effects:
• It may cause mild diarrhea or vomiting in some children.

Vaccines
Vaccine Immunizing Agent Route Adverse Reactions
BCG Live attenuated M.bovis ID Fever, regional
adenitis
Typhoid Whole cell, killed
bacteria
IM Fever ,local swelling
Cholera Inactivatedbacteria SC;IM Fever; swelling
Diphtheria Inactivated toxins IM Local reactions
Tetanus Inactivated toxins IM Local reactions
Typhus
vaccine
Killed organisms SC Allergic reactions

Adjuvants
• An adjuvant is a substance that increase immune response to foreign antigen.
• Adjuvants may be added to a vaccine to modify the immune response by
boosting it such as to give a higher amount of antibodies and a longer-lasting
protection.
• Adjuvants are also used in the production of antibodies from immunized
animals.
• The most commonly used adjuvants include aluminum hydroxide and paraffin
oil.

Adjuvants
• Aluminium Hydroxide
• It is a white gelatinous precipitate in aqueous suspension.
• The mechanism of action includes the formation of a depot at the injection site, which
enabled the slow release of antigen and the stimulation of antibody producing plasma
cells.
• Side effects:
• Pain at the site of injection.
• Fever .

Immunoglobulins
• Immunoglobulins
• Antibody molecule consists of two light and two heavy chains
composed of different domains

Immunoglobulins
• Immunoglobulins
• The Fab fraction serves as the antigen binding site.
• The specific antigen-binding properties of an IgG molecule are conferred by the three
dimensional stearic arrangement inherent in the amino acid sequence of the variable
region of the light and the heavy chains of the molecule.
• This portion of the IgG molecule is called the idiotypic determinant.
• FC fragment is relatively constant and determines the effectors function of the antibody.
• The FC domain is necessary for interaction with complement cascade.

Immunoglobulins
• Human Normal Immunoglobulin:
• Roughly plasma protein can be fractionated into four important components, viz. albumin and the alpha, beta
and gamma globulins.
• The Ig obtained from pooled , human, adult blood is known as immune serum, more selective type of a gamma
globulin against a particular infection obtained from the blood of individuals ,this is called as Hyper Immune
Serum or Human Specific Ig.
• Adverse Reactions:
• It can cause pain at the site of injection
• Allergic reactions can occur
• It may give rise to fever ,flushing, shivering joint pain and nausea.

Immunoglobulins
• Monoclonal antibodies:
• Are the antibodies produced by single clone of B cells, which are now being used
for diagnostic procedures and therapeutic purposes.
• Mechanism of action :
• They block the characteristic of targeted antigen, its function ,its cell surface and
tissue distribution, to produce immune conjugates.

Immunoglobulins
• Adverse reactions:
• Hypersensitivity reactions.
• Activation of inflammatory cells.
• Therapeutic uses of mAb:
• Used as immunosuppressant.
• In auto immune disease.

Miscellaneous agents
• LEVAMISOLE:
• It is available as ergamisol or vermisol.
• First synthesised to treat parasitic worm infections.
• Used as immuno modulating agent in cancer.
• It is given orally, which is rapidly absorbed and it crosses BBB.
• Metabolized in liver and excreted through urine.
• It restores depressed B and T cells functions.

• MOA
• It acts by modulating cell mediated immunity.
• The ganglia in worms are stimulated causing paralysis and expulsion of live worm, by
the activation of macrophages.
• Dose: It is administered in a dose of 50mg thre e times a day for 4-6 weeks.

• Nausea.
• Muscle pain.
• Skin rashes.
• Drowsiness.

• ISOPRINOSINE:
• It is also available as inosine.
• Useful as immunostimulant in immunodefecient patients.
• It is antiviral agent.
• It acts as a immunostimulant, an analog of thymus hormones.
• It is most commonly used to treat the rare measles complication subacute
sclerosing panencephalits.

• Mechansim of action :
• Isoprinosine is a synthetic purine derivative with immunomodulatory and anti viral properties.
• The action of Isoprinosine can be summarized as follows:
• It normalizes the cell-mediated immunity by stimulating the differentiation of T-lymphocytes into
T-helper cells, and increasing production.
• It increases the humoral immune response by stimulating the differentiation of B- lymphocytes into
plasma cells and by enhancing antibody production.
• It inhibits viral growth by suppressing viral RNA synthesis while potentiating depressed lymphocytic
action

Immunosupressants
• Immunosuppression involves an act that reduces the activation or efficacy of the
immune system
• Immunosuppressants are used to control severe manifestations of allergic,
autoimmune and transplant-related diseases
• Now over 80 autoimmune diseases and several common allergic conditions in which
immunosuppressant's are used
• Prevent the rejection of transplanted organs and tissues
• Treatment of autoimmune diseases or diseases that are most likely of autoimmune
origin
• Treatment of some other non-autoimmune inflammatory diseases

Classification of Immunosupressants
• 1.PHYSICAL IMMUNOSUPRESSANTS
• Includes Total Lymphoid Irradiation, Plasmapheresis, thoracic duct drainage
• Inhibits Cell division ,cell activation, Antibody production
• 2.CHEMICAL IMMUNOSUPPRESSANTS:
• I. Corticosteroids
• II. Cytostatics
• III. Antibodies
• IV. Drugs acting on Immunophilins
• 3.BIOLOGICAL IMMUNOSUPPRESSANTS:
interferon's, interleukins, colony-stimulating factors, monoclonal antibodies

Physical Immunosupressants
• Total Lymphoid Irradiation (TLI ):
• Fractionated irradiation focused on Lymphoid tissues, with shielding of Bone
marrow, Lungs ,Non lymphoid tissues
• Induces formation of large granular Lymphocytes lacking T,B & Macrophage
markers which non specifically suppresses Ag –specific cytolytic arm of Allogenic
immune reactions
• TLI can induce true Transplantation tolerance to Renal allografts in humans
• UV-B light is absorbed by skin Urocanic acid & undergoes isomerization to Cis
form which induces suppression through effect on Dendritic APC

• Myelosuppression
• Skin changes
• Nausea and vomiting

• Plasmapheresis:
• Removing plasma hemocomponent that is circulating with pathogens and replacing it with a
suitable solution
• Useful adjunct to chemotherapy for removing circulating immunoglobulins or immunoglobulin
components in multiple myeloma and other dysproteinemias
• Rapidly removes pathogenic antibody
• Must be combined with B lymphotoxic drug to prevent rebound (e.g. cyclophosphamide,
steroids)
• Combination with IV Ig very powerful
• Risks include cardiovascular instability

• Thoracic duct drainage:
• Woodruff demonstrated that synergism of thoracic-duct drainage with
lymphoid-depleting modality, antilymphocyte serum
• Effective and safe in decreasing the immunologic response of the recipient of
renal transplants from genetically related donors
• Lymphocytapheresis using TDD is very selective for removing lymphocytes
(especially helper T- cells)

Chemical Immunosupressants
• Corticosteroids:
• Prednisone , Prednisolone Dexamethasone,Methylpredinsolone
• They have both anti-inflammatory action and immunosuppressant effects
• Mechanism of action:
• bind to glucocorticoid receptors and the complex interacts with DNA to inhibit
gene transcription of inflammatory genes
• stimulates migration of T cells from intravascular tissue to lymph nodes
• Inhibit mitosis of lymphocytes

• Reduce size and lymphoid content of the lymph node and spleen
• Inhibit the production of inflammatory mediators, including PAF, leukotrienes,
prostaglandins, histamine and bradykinin
• Decrease production of cytokines IL-1, IL-2, interferon, TNF

• Cytostatics
• Cytostatics inhibit cell division
• In immunotherapy, they are used in smaller doses than in the treatment of
malignant diseases.
• They affect the proliferation of both T cells and B cells.
• Due to their highest effectiveness, purine analogs are most frequently
administered.
• It includes the following: Alkylating agents; Antimetabolites

• 1. Alkylating agents:
• The alkylating agents used in immunotherapy are nitrogen mustards
(cyclophosphamide), nitrosoureas, platinum compounds, and others
• In small doses, it is very efficient in the therapy of systemic lupus erythematosus,
autoimmune hemolytic anemias, Wegener's granulomatosis and other immune
diseases

• Cyclophosphamide
• Cyclophosphamide is an alkylating agent. It is a widely used as a cytotoxic agent.
• It is given orally as well as intravenously with efficacy
• Mechanism of action: suppress bone marrow function
• It is inactive in parent form, and must be activated to cytotoxic form by liver CYT450 liver
microsomal system to 4‐Hydroxycyclophamide and Aldophosphamide. 4‐Hydroxycyclophamide
and Aldophosphamide are delivered to the dividing normal and tumor cells.
• Aldophosphamide is converted into acrolein and phosphoramide mustard. They crosslink DNAs
resulting in inhibition of DNA synthesis

• Side effects: Usually large Doses of cyclophosphamide is associated with ‐
• a. Pancytopenia
• b. Hemorrhagic cystitis
• c. Nausea and vomiting
• d. Cardiac toxicity
• e. Electrolyte imbalances

• Antimetabolites
• Includes folic acid analogues, such as methotrexate; purine analogues such as
azathioprine and mercaptopurine pyrimidine analogues; protein synthesis inhibitors
• Azathioprine :
• Prodrug that releases 6-mercaptopurine
• Mechanism of Action:
• Converts 6-mercaptopurine to tissue inhibitor of metalloproteinase, which is converted
to thioguanine nucleotides that interfere with DNA synthesis; thioguanine derivatives
may inhibit purine synthesis

• Uses:
• a. Used for graft rejection
• b. Normally used in combination with corticosteroids.
• Side effects:
• Bone marrow suppression (leukopenia, anemia), Skin rashes,nausea
• Liver toxicity ,macrocytosis

• Mycophenolate mofetil
• Mycophenolic acid from penicillium molds
• Prevents T- and B-cell proliferation by inhibition of de novo purine synthesis by
inhibition of inosine monophosphate dehydrogenase
• Dosage 1 to 2 g/day in divided doses
• CLINICAL USE:
• Solid organ transplants for refractory rejection.
• Steroid-refractory hematopoietic stem cell transplant patients.

• Combined with prednisone as alternative to CSA or tacrolimus.
• Rheumatoid arthritis, & dermatologic disorders.
• Leukopenia, neutropenia.
• Lymphoma
• GIT toxicity

Antibodies
• block T cell surface molecules involved in signaling immunoglobulins
• They are of two types:
• Polyclonal antibodies & Monoclonal antibodies
• Polyclonal antibodies:
• Obtained from plasma or serum of horses hyper-immunized with human lymphocytes.
• Inhibit T lymphocytes and cause their lysis, which is both complement mediated cytolysis and
cell-mediated opsonization followed by removal of reticuloendothelial cells from the circulation
in the spleen and liver.
• Antithymocyte globulin (ATG)
• Antilymphocyte globulin (ATGAM)

Antibodies
• Polyclonal antibodies
• agents contain antibodies specific for many common T cell antigens including CD2, CD3, CD4,
CD8, CD11a, CD18
• Blocks T-cell membrane proteins (CD2,CD3, CD45, and so forth), causing altered function, lysis,
and prolonged T-cell depletion
• CLINICAL USE:
• Combined with cyclosporine for bone marrow transplantation.
• To treat acute allograft rejection.
• Steroid-resistant rejection.

Antibodies
• Leukopenia ,Thrombocytopenia
• serum sickness
• muscle pain
• lymphopenia

Antibodies
• Monoclonal antibodies are antigen-specific immunosuppressants that will reduce immune response to alloantigens of
the graft while preserving the response to alloantigens to unrelated antigens
• Early rejection prophylaxis and treatment of rejection.
• Muromonab-CD3 (OKT3):
• Directed against CD3 component of T-cell–receptor signal-transduction complex
• Binds to CD3 associated with T-cell receptor,leading to initial activation and cytokine release, followed by blockade of
function, lysis, and T-cell depletion
• Severe cytokine-release syndrome, pulmonary edema, acute renal failure, gastrointestinal disturbances, changes in
central nervous system

Drugs acting on immunophilins
• Cyclosporine:
• 11-amino-acid cyclic peptide from Tolypocladium inflatum
• Binds to cyclophilin intracellular protein receptors complex inhibits calcineurin
phosphatase and T-cell activation
• CLINICAL USE:
• Kidney, liver, heart organ transplantation used in combination with azathioprine

Drugs acting on Immunophilins
• Nephrotoxicity, hemolytic–uremic syndrome, hypertension
• neurotoxicity, gum hyperplasia
• skin changes ,hirsutism,
• post-transplantation diabetes mellitus
• hyperlipidemia

New immunosuppressive drugs
• FTY 720
• derived from myriocin, a fungus-derived sphingosine analogue
• Works as an antagonist for sphingosine-1-phosphate receptors on lymphocytes,
enhancing homing to lymphoid tissues and preventing egress, causing
lymphopenia

• Prescribed for
• Renal transplant
• Multiple sclerosis
• Side effect
• Reversible first-dose bradycardia, potentiated by general anesthetics and beta-
blockers
• nausea, vomiting, diarrhea, increased liver-enzyme

• Etanercept (Enbrel)
• Recombinant DNA drug
• binds TNF (tumor necrosis factor) in the circulation and in the joint, preventing
interaction with cell surface TNF receptors thereby reducing TNF activity
• Subcutaneous injection

• Side effects
• Susceptibility to opportunistic infection
• ISA 247
• novel isomeric cyclosporine A analog mixture a calcineurin inhibitor.
• treatment of psoriasis and prevention of organ rejection after transplantation

General method of the preparation of
bacterial vaccines

General method of the preparation of
bacterial vaccines
Dr. LAKSHMI
ASSISTANT PROFESSOR
GHAZIABAD

Vaccines
• Definition: Vaccine is a preparation/suspension or extract of a
dead/attenuated (weakened) germs of a disease which on inoculation
(injection) into a healthy person provides temporary/permanent,
active/passive immunity by inducing antibodies formation.
• Thus antibody provoking agents are called vaccines.
• Vaccines may be prepared from one species only or from a mixture of
two or more species. The process of introduction of vaccine into an
individual to provide protection against a disease is called vaccination.

Vaccines
• Vaccination and immunization are two different processes.
• Vaccination only refers to the administration of a vaccine or toxoid, while
immunization is the process by which the body produces antibodies against
the vaccine preventable diseases through administration of specific
vaccines.

Types of Vaccines
• Vaccines are of three types:
• (i) Killed (inactivated) vaccines
• (ii) Live attenuated vaccines
• (iii) Toxoids

Types of Vaccines
• Vaccines are of three types:
• (i) Killed (inactivated) vaccines:
• consist of microorganisms killed by heat or chemicals.
• (ii) Live attenuated vaccines:
• Consist of live bacteria or viruses which have been rendered avirulent. They nevertheless
grow and multiply in the body of the host to a limited extent.
• In individuals with impaired host defence, e.g.
• (a) Leukaemia or other malignancies, especially those receiving cytotoxic chemotherapy.
• (b) Systemic lupus erythematosus.

Types of Vaccines
• (c) Corticosteroid recipients.
• (d) AIDS and other immune deficiency states.
• The limited virulence of organisms in the live vaccine may be
sufficient to cause a disease; live vaccines are contraindicated in
them. Two live vaccines, if not given together, should preferably be
administered with a gap of 1 month.
• (iii) Toxoids: are modified bacterial exotoxins so that toxicity is lost but
antigenicity is retained. The term 'vaccine' is sometimes restricted to
preparations of whole microorganisms and toxoids are enumerated

Production of Vaccines
• Standard manufacture uses a bacterial or viral antigen, e.g.
bacterium or virus, which may be killed or may be living but
attenuated.
• To make a live attenuated vaccine, the disease-causing organism is
grown under special laboratory conditions that cause it to lose its
virulence or disease-causing properties.
• The attenuation can be obtained by heat or by passage of the virus in
foreign host such as embryonated eggs or tissue culture cells.

Production of Vaccines
• Cell cultures are required for viral vaccines since viruses can replicate
only inside the living cells.
• For example To produce the Sabin polio vaccine, attenuation was only
achieved with high inocula and rapid passage in primary monkey kidney cells.
• Inactivated vaccines are produced by killing the disease-causing
microorganism with chemicals or heat.

Pathogen(Seed or Clinical isolate)
Culture Attenuation Cloning,GMO
Ag
Purification
Inactivation
VACCINE
Seed(Live
attenuated)
Culture
VACCINE
Seed
Culture
VACCINE
Inactivation
VACCINE
Purification
VACCINE
wP,
HAV
Rab, Flu
MMR,OPV HBV,HPV
aP

SELECTING THE STRAINS FOR
VACCINE PRODUCTION
GROWING THE MICRO-
ORGANISMS
ISOLATION & PURIFICATION OF
MICROORGANISM
INACTIVATION OF ORGANISM
FORMULATION OFV
ACCINE
QUALITY CONTROLAND LOT RELEASE
UPSTREAM
PROCESSING
DOWNSTREAM
PROCESSING

Selecting the strains for vaccine production
• The Seed(Strain) -
• Manufacturing begins with small amounts of a specific virus (seed).
• Viruses or Bacteria used in manufacture shall be derived from a Seed Lot System.
• The virus must be free of impurities, including other similar viruses and even
variations of the same type of virus.
• The seed must be kept under "ideal" conditions, usually frozen, that prevent the
virus from becoming either stronger or weaker than desired.
• Stored in small glass or plastic containers.

• The choice of the seed is depends on a number of factors including the
efficacy of the resulting vaccine, and its secondary effects.
• If possible, the bacterial strain or cell line should be obtained from a
recognized culture collection with an established and documented
provenance.
• Alternatively, if the chosen vaccine strain is an “in house” clinical isolate, it
will be necessary to compile a complete history of the strain, including
details of its isolation, identification, and maintenance for product
registration.
Selecting the strains for vaccine production

Growing microorganisms
• Growing Bacteria
• Methods used are :
• Batch culture
• the microbe is grown in a closed vessel
• typically in a test tube or flask
• Continuous culture
• the microbe is grown in vessel which has medium constantly
added and spent medium constantly removed.

• Growing Viruses
• Methods used are :
• Cell (tissue) cultures – cultured cells grow in sheets that support viral
replication and permit observation for cytopathic effect.
• Bird embryos – incubating egg is an ideal system; virus is injected
through the shell.
• Live animal inoculation – occasionally used when necessary
• Transgenic animals
Growing microorganisms

Isolation and purification
• Product isolation is the removal of those components whose properties vary
markedly from that of the desired product.
• Purification selectively separates and retains the desired product at the highest
purity per its pre-determined specification. (Remove unwanted compounds)
• The most common method of vaccine production is based on an initial
fermentation process followed by purification.
• Centrifugation
• Filtration
• Chromatography

Inactivation
• Killed/Inactivated Vaccine
• Virus inactivation:
• Viruses can be lipid-coated(enveloped) or non-enveloped.
• Virus inactivation involves dismantling a virus’s ability to infect cells without actually eliminating
the virus.
• Virus inactivation works by one of the following mechanisms:
• By attacking the viral envelope or capsid and destroying its ability to infect or interact with
cells.
• By disrupting the viral DNA or RNA and preventing replication.

Inactivation
• Other methods
• Solvent/detergent (S/D) inactivation
• Pasteurization
• Acidic pH inactivation(Low pH Treatment)
• Ultraviolet (UV) inactivation

Formulation of Vaccine
Suspending fluids –
• The liquid which contains the chemicals used during production which kill or weaken
the organism for use in vaccines.
• Sterile water, saline or fluids containing protein,
• Egg proteins are found in influenza and yellow fever vaccines, which are prepared using
chicken eggs
• Yeast Proteins, Hepatitis B vaccines are made by transfecting cells of Saccharomyces
cerevisiae (baker’s yeast) with the gene that encodes hepatitis B surface antigen, and
residual quantities of yeast proteins are contained in the final product.

• Preservatives and stabilizers
• Albumin, Phenols, Glycine
• Monosodium glutamate (MSG) and 2-phenoxy-ethanol which are used as stabilizers in a few vaccines to help the vaccine remain
unchanged when the vaccine is exposed to heat, light, acidity, or humidity.
• Antibiotics , which are added to some vaccines to prevent the growth of bacteria during production and storage of the vaccine.
• Antibiotics that are used during vaccine manufacture include neomycin, streptomycin, polymyxin B, chlortetracyline, and
amphotericin B.
• Thimerosal is a mercury-containing preservative that is added to vials of vaccine that contain more than one dose to prevent
contamination and growth of potentially harmful bacteria. Eg. diphtheria-tetanus-acellular pertussis (DTaP), hepatitis B, and
Haemophilus influenza type B (Hib).

• Inactivating Agents-
• Formaldehyde is used to inactivate bacterial products for toxoid vaccines, (these are vaccines that use an
inactive bacterial toxin to produce immunity.)
• It is also used to kill unwanted viruses and bacteria that might contaminate the vaccine during production.
• Most formaldehyde is removed from the vaccine before it is packaged.
• It is used to inactivate influenza virus, poliovirus, and diphtheria and tetanus toxins.
• β-propiolactone, which is used to inactivate rabies virus
• Glutaraldehyde, which is used to inactivate toxins contained in acellular pertussis vaccines.

• Adjuvants or enhancers –
• Aluminum gels or salts (Alum)
• Alum is used in several licensed vaccines including:
• diphtheria-pertussis-tetanus
• diphtheria-tetanus(DT)
• DT combined with Hepatitis B (HBV)
• Haemophilus influenza B

• Inactivated polio virus
• Hepatitis A (HAV)
• Streptoccucus pneumonia vaccine
• Meningococccal vaccine
• Human papilloma virus (HPV)

45
Cell culture Harvest Bulk Purification
Formulation
Filling
Labeling
Packaging
virus
(production
seed)



Add

Inoculation
cell
Adjuvant Stabilizer
Bulking agent
Preservative
Inspection
centrifugation
filtering
How to produce Vaccines

After incubation the egg
white contains millions of
vaccine viruses which are
harvested and then
separated from the egg
white.
Vaccine
virus
multiplied
Virus is spun to
separate it from
the egg white
Vaccine
virus
The vaccine virus is
injected into a 9 to 12
day old fertilized egg and
incubated for 2 to 3
days.(during this time the
virus multiplies)

Other immunological products
• In-vivo Diagnostics
• They are used to demonstrate an Immunogenic response like previous exposure
to a pathogen.
• Helpful in the diagnosis of diseases.
• Examples: Tuberculin, Mallein, Histoplasmin, Coccidiodin, Brucellin

• Immune Sera
• To prepare an immune serum, horses or other animals are injected with a
sequence of spaced doses of an antigen until a trial blood sample shows that the
injections have induced a high titre of antibody to the injected antigen. An
adjuvant may be used if required.
• The animals must be in good health, free of infections and from sources free of
TSEs, and kept under veterinary supervision

• Preparation

• Human Immunoglobulins
• Source: Human immunoglobulins are preparations of the immunoglobulins,
principally (IgG) subclasses, that are present in human blood. They are derived
from the plasma of donated blood and from plasma obtained by plasmapheresis.
• Specific immunoglobulins, that is immunoglobulins with a high titre of a particular
antibody, are usually prepared from smaller pools of plasma obtained from
individuals who have suffered recent infections or who have undergone recent
immunization and who thus have a high titre of a particular antibody.

• Monoclonal Antibodies

Storage conditions and stability of official
vaccines
• Overview
• Vaccine Storage Basics
• Training
• Maintaining Temperatures
• Storage & Handling
• Multi-dose Vials

vaccines
• Vaccine Storage – The Basics
• Designated and trained staff
• Adhering to protocols, guidelines and standard operating procedures in the practice
setting
• Maintain vaccine potency and minimize waste
• Maintain required fridge temperatures
• Organization and placement of vaccine supply and maintain optimal handling practices
• Contingency planning for refrigerator malfunctions and electricity disruptions

vaccines
• Designated Staff & Staff Training
• Health care providers storing and handling publicly-funded vaccines require
knowledge:
• Importance of cold chain
• How to recognize a cold chain incident
• The appropriate action to be taken in the event of a vaccine exposure
• Vaccine storage and handling practices

vaccines
• Maintaining Potency of Vaccines
• Always store between +2 ͦ C and +8 ͦ C
• Notify NWHU (Northwestern health university) of vaccine exposures outside
recommended storage temps
• Avoid freezing of vaccines and diluents
• Keep vaccine/diluents under cold chain, remove and prepare right before
administration

vaccines
• Maintaining Temperatures
• Know functions and components of the vaccine refrigerator
• Modify, stabilize and maintain temperature between +2 and +8 ͦC
• Ensure refrigerator capacity
• Keep the door tightly closed and reduce number of openings
• Ensure the refrigerator is optimally placed

vaccines
• Be aware of seasonal variations in room temperature
• Perform regular maintenance
• Prevent accidental shut off or unplugging
• Fill drawers, lower shelves, and door with water bottles
• Keep ice packs in the freezer for use during transport or refrigerator malfunction
• Have a contingency plan

vaccines
• Storage and Handling Vaccine must be kept secured
• Insulated vaccine container(s) with packing material and a
temperature monitoring device is on hand

vaccines
• Storage and Handling Cold chain materials available:
• How to monitor refrigerator temperature magnet
• Protect your vaccine, Protect your patients poster
• Vaccine Storage and Handling Guidelines

vaccines
• Storage and Handling Store in the middle of internal shelves
• Organize vaccine by product, and store in original packaging
• Leave space between vaccine products and packages
• Check expiry dates regularly – never store expired vaccine
• Rotate Stock

vaccines
• Storage and Handling
• Protect vaccines from sunlight and fluorescent light
• Use vaccine previously exposed to a cold chain incident first (if
cleared)
• Remove vaccine from refrigerator right before immediate use

vaccines
• Storage and Handling
• Never leave vaccine on the counter or floor
• Do not store anything other than vaccines and medications that
require refrigeration in the vaccine storage unit

vaccines
• Multi-dose Vials Mark/date the vial when it was opened
• Return unused vaccine to the refrigerator immediately after drawing up the
required dose
• Aseptic technique for withdrawal of vaccine must be followed at all times
• Discard within 30 days or as per the manufacturer’s instructions indicated
in the product monograph

vaccines
• Stability of vaccines: key definitions
• Stability is the ability of a vaccine to retain its chemical, physical,
microbiological and biological properties within specified limits throughout
its shelf-life.
• Real-time/ real condition stability studies: physico-chemical, biological &
other vaccine characteristics during & up to the expected shelf-life and
storage periods under expected handling and storage conditions

vaccines
• What is a Stable Vaccine?
• A vaccine with a very long shelf life
• A vaccine lot that complies with release specifications throughout shelf life
• Technical specifications: Vaccine that complies with the principles of the
initial license through out life cycle Technical, non-clinical and clinical
specifications

vaccines
• Factors Having a Strong Impact on Stability of Vaccines
• Purity
• Formulation
• Stabilizers: Human Serum Albumin (HAS), recombinant human Albumin (rHA),
Gelatine, Sugars, Sorbitol Thiomersal AlOH, AlPO Ions (Buffer systems)
• Pharmaceutical form: Lyophylized versus liquid
• Storage: Frozen versus refrigerator

vaccines
• Guidelines on Stability of Vaccines
• ICH harmonised tripartite guideline quality of biotechnological products:
• Stability testing of biotechnological/biological products, q5c, dated 30 november 1995
• Development of a CPMP Points to Consider on Stability and Traceability Requirements for Vaccine
Intermediates, dated 2000
• WHO guideline on stability evaluation of vaccines, dated 2006
• Other regional guidance All these guidance documents provide rather general instructions.
Product specific cases must be considered case by case.

vaccines
• Product Specific Issues
• To what extend may critical quality attributes change during shelf life without affecting safety and efficacy?
• Are all potential changes detectable by technical means?
• How many stability lots are needed to address the impact on stability due to variances inherent to the
manufacturing process
• Wide specifications
• Number and age of intermediates
• How can these uncertainties best be investigated during clinical development and post-marketing
monitoring

vaccines
• Stability evaluation: guiding principles
• Vital part of quality and safety assessment of a vaccine
• Temperature sensitivity
• Specific biological activity: potency assay
• Design of stability studies - depends of the objectives of stability studies:
• 1) Determine shelf-life, storage conditions and to support licensing
• 2) monitor vaccine stability in the post-licensure period
• 3) Support manufacturing changes: comparability
• Defining acceptance criteria for stability parameters by clinical trials

vaccines
• The vaccine life cycle
• Registration/launch is the critical time: studies prior to that point are
aimed at obtaining information to support registration.
• Subsequent studies are aimed at assuring that the assessments at the
time of registration are still correct

vaccines
• Using stability data and specifications to set shelf life
• Goal: Throughout its shelf life, product must be comparable to batches
shown to be safe and effective in clinical studies
• Stability data are used to make predictions that can be extrapolated to
future batches of product
• The most accurate predictions are based on mathematical modeling of
biologically relevant stability-indicating parameters

vaccines
• Potency Definition
• Specific ability or capacity of the product, as indicated by appropriate
laboratory tests or by adequately controlled clinical data obtained
through the administration of the product in the manner intended, to
effect a given result

vaccines
• Why do we do a potency assay?
• In development, to Assure that safe potencies are not exceeded in clinical trials Obtain
information that will support licensure-including correlation of potency with clinical response
• After licensure, to assure that lots behave similarly to those tested in the clinical trials that
supported licensure
• The potency should not be below the lowest potency believed to be efficacious The potency
should not exceed the highest potency believed to be safe
• The potency assay thus provides a “bridge” between licensed material and the clinical trials

vaccines
• What does a potency assay tell us?
• The assay estimates the mean potency value for a lot
• There are two sources of variability in the measured potency of an individual vial from
the same vaccine lot– manufacturing variability and assay uncertainty
• Thus, we can never know the actual potency in an individual vial that was used in a
clinical trial
• We can know the characteristics of the lot of vaccine from which that vial came (we
routinely estimate the mean potency)

vaccines
• Variability in potency assay results
• The standard error of the mean potency of a lot provides information about the accuracy
with which we can estimate the mean potency, and thus provides information about
both of these sources of variability, combined
• The SEM provides information about our level of certainty that we know the mean
potency of the lot; just as the mean potency does not provide information about
individual vials (which is impossible to obtain, when assays are variable), the SEM
provides information about that estimate

vaccines
• Variability in potency assay results
• The standard deviation of the mean potency provides information about
the distribution of potency test values, but does not tell us about the
underlying actual values
• Conformance to GMP and assuring consistency of manufacture provides
assurance that the manufacturing variability remains constant over time

vaccines
• Clinically relevant parameters
• L.L.: the lowest dose at which we are comfortable, based on the clinical
data, that the vaccine is going to be effective. This is normally a
specification.
• U.L.: The highest dose at which we are comfortable, based on the clinical
data, that the vaccine is going to be safe. This is normally a specification.

vaccines
• Dose
• Stability profile
• Analytical assay variability
• Confidence limits

vaccines
• Two models
• Compliance model –
• Substantially all potency measurements should exceed some predefined quantity
• Often, the mean potency from clinical trials is used to set this minimum specification
• This is very difficult (perhaps impossible) to reliably implements in original clinical tests were performed
based on an understanding of mean potency, not on an understanding of “all potency measurements”
• Estimation model – The mean potency of substantially all vaccine lots should be at a level that is not less
than the mean potency of vaccine lots shown to be effective in clinical trials

vaccines
• Compliance model
• Shelf life is set arbitrarily depending on the luck associated with each
individual assay at each time point, and based on the starting potencies of
lots on test
• Release potency must be high enough to prevent test results that fall below
L.L. due to assay variability, from requiring an unacceptably short shelf life,
or from subsequently failing stability monitoring studies

vaccines
• Estimation method
• Statistical methods (when appropriate, a regression line) also can be
used to estimate lower bounds on the mean potency estimate at end-
expiry for any given release potency, thus allowing estimation of shelf
life

vaccines
• Development-what do we need to know about stability
• What are the kinetics of decay?
• What are the degradation products?
• Are there stability-influencing factors that should also be controlled?
• Do we have sufficient knowledge of the stability of intermediates?

vaccines
• Are the assays adequate?
• – Are potency assays stability-indicating?
• – Are potency assays precise enough to support product development?
• Do we have a sufficient understanding of material tested in clinical trials?
• Do we have a sufficient understanding of product performance at potency
ranges likely to be encountered post-licensure?

vaccines
• TYPES OF STABILITY
• Real Time Stability : Long term stability under recommended storage
stability under recommended storage conditions for the shelf life proposed
for the product.
• Accelerated Testing : Studies under exaggerated storage conditions.
• Stress studies: More severe conditions than those used for accelerated
studies.

vaccines
• TEMPERATURES
• Real Time: Storage Temperature of the product e.g. 2-8°C for Tetanus
Vaccine
• Accelerated 15±2°C higher than the maximum temperature of
storage at real time storage of the product e.g. 25 ± 2°C
• Stress: +10°C ±2°C than the accelerated temperature e.g.35 ± 2°C

vaccines
• Samples
• Drug substances:
• Intermediate product: active raw material
• Formulated bulk: final bulk
• Drug product:
• Finished product: final lot

vaccines

vaccines
• Designing
• Bracketing: Extremes of certain design factors e.g.
• Strength: 10 ug, 25ug, 100 ug
• Package size: 1 dose, 5 dose ,10 dose
• Matrixing: A selected subset of the total number of the possible samples
for all factors is tested at specified time and at a subsequent time another
subset of samples for all factors is tested.

vaccines
• Stability indicating parameters
• Choice of parameters to be tested: depends of the product characteristics
• Link between vaccine quality and efficacy or safety as demonstrated in clinical
trials – ideal case
• Potency, antigen content,appearance, pH, completeness of adsorption, adjuvant
content; physicochemical properties etc
• New vaccines: parameters to be identified during the product development

vaccines
• Real Time 2-8°C for 4 years (for 3 years expiry)
0,3M,6M,9M,12M,18M,24M,36M & 48 M
• Accelerated Temperature 20-25°C for 6 months 0,1M,2M,3M & 6
Months
• Stress Study: 35°C for 1-2 months 0,1,2,3,4,5,6,7& 8 weeks

vaccines
• Acceptance criteria
• In general, significant change is defined as:
• 5% change in assay from the initial value or failure to meet the acceptance criteria by
biological or immunological parameters or as applicable for the specification of the product.
• Any degradation product (impurity) exceeding its acceptance criteria
• Failure to meet the acceptance criteria for physical and chemical parameters
• Failure to meet the acceptance criteria for dissolution for stipulated dosages.

vaccines
• Cumulative age of an antigen in the final Product
• Reported as a major issue in practice
• Final product should be stable during the whole period of shelf-life irrespective of the
age of its intermediate products
• Data should be collected on an ongoing basis – usually not available before approval of
storage periods and shelf-life
• Accelerated stability studies - to demonstrate that an aged intermediate did not affect
stability of the final product

vaccines
• Thermal stability testing – lot release
• An indicator of consistency of production
• Not designed to provide a predictive value of real time
• TS is a shelf-life specification in current WHO vaccine specific
recommendations
• Live attenuated vaccines – OPV, MMR, YF
• For other vaccines: consider relevance of the rate of change for safety and
efficacy

vaccines
• Stability of combined vaccines
• Determination of the shelf-life should be based on the shortest shelf-life
component
• Data obtained for monovalent vaccines should support stability of
combined vaccine
• Stability of combined vaccines should not be based on the extrapolation of
the stability data of the individual components alone

vaccines
• Labelling
• Recommended storage conditions and expiry date
• Sensitivity of vaccines to environmental factors (eg light,freezing) and preventive
measures
• If VVMs are to be used, adequate stability data should be generated to support
selection of appropriate VVM for the vaccine in question

Hybridoma technology
Dr. LAKSHMI
ASSISTANT PROFESSOR
GHAZIABAD

Hybridoma Technology
• Hybridomas are cells formed via fusion between a short-lived
antibody-producing B cell and an immortal myeloma cell.
• Each hybridoma constitutively expresses a large amount of one specific
mAb, and favored hybridoma cell lines can be cryopreserved for long-
lasting mAb production.

History:
• In 1975, this technology developed by
• Georges J.F.Kohler and Cesar Milstein.
• And in 1984, they shared a Nobel prize for this discovery.
• They make a hybrid cell that will make a numbers of monoclonal antibodies
against antigen .

Principle:
• The hybrid cell has the capacity of antibody production derived from B-cells
(spleen cell ).
• At the same time it can divide continuously by the quality derived from
myeloma cell.
• By combining the desired qualities of both the cells, the technology ensures
large, antibody production of single specificity.
• Specific hybridomas(spleen cell and myeloma cell) obtain monoclonal
antibodies in artificial media, this technology called as HYBRIDOMA
TECHNOLOGY.

Monoclonal antibody:
• Monoclonal antibodies (mAb) are antibodies that are identical
because they are produced by one type of immune cell, all clones
of a single parent cell.
• Basically produced by white blood cell which is called as plasma
cell.
• Is used for treatment of cancerous cells and as anti-venom( anti
snake venom)

Procedure:
• Immunization of specific animal which generate hybridoma cell with
spleen cell.
• Isolation of myeloma cells.
• Fusion between spleen cell and myeloma cell.
• Selection of HAT medium.
• Isolation of hybridoma cell.
• Screening of hybridoma cell.

1. Immunization of specific animal.
• An antigen immunized to an animal (like mice) via intravenously (directly to
blood) by injection.
• Where in spleen it activate B-cell which produce plasma cell (spleen cell).
• Plasma cell to produce monoclonal antibodies
• Isolation of plasma cell from spleen of animal.

2. Isolation of myeloma cells.
• Myeloma cells are cancerous cells which is isolated from bone-marrow.
• Myeloma cells are generally immortal in nature (that which never dies) and has
multiplication property.

3. Fusion of spleen cell and myeloma cell.
• It requires PEG (poly ethylene glycol) medium for fusion
• It can also done by electro fusion.
• Fusion between spleen cell and myeloma cell produced five different types of cells.
• Fused plasma
• Fused myeloma
• Hybridoma
• Unfused plasma
• Unfused myeloma

•4. Selection of HAT medium.
( Hypoxanthine, Aminopterin, Thymidine)
• Before multiplication of Anti-body, it has to synthesize new copy of DNA
and for that it require synthesis of nucleotide.
• For synthesis of nucleotide mainly two pathways are there:
• Salvage pathway
• De-novo Synthesis
• In 1 , Salvage pathway it requires degraded part of old nucleotide to
produce new nucleotide.
• In 2, De-novo synthesis it synthesized completely new nucleotide by
small molecules (sugar, amino-acid).

• So in HAT medium, Cells not synthesized by De-novo synthesis due to presence
of Aminopterin in HAT medium which blocks Di-hydro follate enzyme which is
necessary for these synthesis.
• For synthesis in salvage pathway it must requires HGPRT enzyme
(Hypoxanthine Guanine Phospho-Ribosyl Transferase).
• Where hypoxanthine and thymidine are used as precursors.
4. Selection of HAT medium.

5. Isolation of hybridoma cell
Myeloma cell doesn’t
have HGPRT enzyme
Spleen cell have
HGPRT enzyme
1. Fused plasma
2. Fused myeloma
3. Hybridoma
4. Unfused plasma
5. Unfused myeloma
HGPRT
present
absent
present
present
absent

• Fused myeloma and unfused myeloma didn’t have HGPRT enzyme so,
can’t survive in HAT medium.
• Fused plasma and unfused plasma have HGPRT enzyme but didn’t
have long-life.
• Hybrid cell has HGPRT enzyme from spleen cell as well as they have
the ability to multiply repeatedly as myeloma cell.
• So, isolation of hybrid cell because is only cell which survive in HAT
medium.
4. Selection of HAT medium.

6. Screening of hybridoma cell.
• ELISA screening method which done by incubating hybridoma culture in which
secondary enzyme gets conjugate and formation of colored product shows
positive hybridoma.
• Used for multiplying the hybridoma cells
• In-vivo
• In-vitro

6. Screening of hybridoma cell.
• In-vivo procedure involves introduction of hybridoma cells into the peritoneal
cavity of the animal , then from ascetic fluid antibodies are isolated.
• In-vitro method involves culturing of hybridoma cells in suitable culture media
and then antibodies are isolated and purified.
• Once a hybridoma colony is established, it will continually grow in culture
medium like RPMI-1640 and produce antibodies.
• Storage: liquid nitrogen.

APPLICATION OF HYBRIDOMA TECHNOLOGY
• Serological:
• Identification of ABO blood group
• Diagnosis:
• Detection of pregnancy by assaying of hormones with monoclonal.
• Separation of one substance from a mixture of very similar molecules.
• Immunopurification:
• Purification of individual interferon using monoclonal.
• Inactivation of T-lymphocytes responsible for rejection of organ transplants.

APPLICATION OF HYBRIDOMA TECHNOLOGY
• Therapy:
• Removal of tumor cell from bone marrow.
• Treatment of acute renal failure.
• Treatment malignant leukemic cells, B cell lymphomas, and a variety of allograft
rejections after transplantation.

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