B Pharmacy 6th sem.

Subject: Pharmaceutical Biotechnology

Subject Code: BP605T

INTRODUCTION TO
BIOTECHNOLOGY
Prepared by: Chamanpreet Kaur
Assistant Professor ( PHARMACEUTICS)
ASBASJSM COLLEGE OF PHARMACY, BELA.
Objective of Course
• Understanding the principle of biotechnology and
importance of immobilized enzymes in pharamaceutical
industries.
• Genetic engineering application in relation to production
of pharmaceuticals

• Learning Outcomes
• Students will learn about biotechnology with references to
pharmaceutical sciences.
• They will learn about biosensor and various methods used
for immobilization of enzymes.
• They will be able to implement the theoretical concept of
genetic engineering and protein engineering.
Module-1
 Brief introduction to Biotechnology with
reference to Pharmaceutical Sciences.
 Enzyme Biotechnology- Methods of
enzyme immobilization and applications.
 Biosensors- Working and applications of
biosensors in Pharmaceutical Industries.
 Brief introduction to Protein Engineering.
 Basic principles of genetic engineering.
 What is biotechnology?
• Biotechnology = bios (life) + logos (study of or
essence)
– Literally ‘the study of tools from living things’

• CLASSIC: The word "biotechnology" was first used in

1917 to describe processes using living organisms to
make a product or run a process, such as industrial
fermentations. (Robert Bud, The Uses of Life: A
History of Biotechnology)

• LAYMAN: Biotechnology began when humans began

to plant their own crops, domesticate animals,
ferment juice into wine, make cheese, and leaven
bread (AccesExcellence)
 What are the stages of biotechnology?

• Ancient Biotechnology
• early history as related to food and shelter,
including domestication

• Classical Biotechnology
• built on ancient biotechnology
• fermentation promoted food production
• medicine

• Modern Biotechnology
• manipulates genetic information in organism
• genetic engineering
 Modern biotechnology
• Cell biology
• Structure, organization and reproduction

• Biochemistry
• Synthesis of organic compounds
•Cell extracts for fermentation (enzymes
versus whole cells)

• Genetics
• Resurrection of Gregor Mendel’s findings 1866
1900s
•Theory of Inheritance (ratios dependent on traits of
parents)
• Theory of Transmission factors

• W.H. Sutton – 1902

• Chromosomes = inheritance factors
 Modern biotechnology

Molecular Biology

• Beadle and Tatum (Neurospora crassa)

• One gene, one enzyme hypothesis
• Charles Yanofsky colinearity
between mutations in genes and amino
acid sequence (E. coli)
• Genes determine structure of proteins

• Hershey and Chase – 1952

• T2 bacteriophage – 32P DNA, not 35S protein
is the material that encodes genetic
information
 Modern biotechnology

• Watson, Crick, Franklin and Wilkins (1953)

• X-ray crystallography
• 1962 – Nobel Prize awarded to three men
• Chargaff – DNA base ratios
• Structural model of DNA developed

• DNA Revolution – Promise and Controversy!!!

• Scientific foundation of modern biotechnology

•based on knowledge of DNA, its replication,
repair and use of enzymes to carry out in vitro
splicing DNA fragments
 What are the areas of biotechnology?

• Organismic biotechnology
• uses intact organisms and does not alter genetic
material

• Molecular Biotechnology
• alters genetic makeup to achieve specific goals

Transgenic organism: an organism with artificially

altered genetic material
 What are the benefits of
biotechnology?
• Medicine
• human
• veterinary
• biopharming

• Environment
• Agriculture
• Food products
• Industry and manufacturing
What are the applications of biotechnology?

• Production of new and improved crops/foods,

industrial chemicals, pharmaceuticals and livestock
• Diagnostics for detecting genetic diseases
• Gene therapy (e.g. ADA, CF)
• Vaccine development (recombinant vaccines)
• Environmental restoration
• Protection of endangered species
• Conservation biology
• Bioremediation
• Forensic applications
• Food processing (cheese, beer)
 Transfer of new Anti-cancer drugs
Culture of plants
genes into animal from single cells Diagnostics
organisms

Cell Monoclonal
Culture Antibodies
Crime solving
Molecular
Biology

DNA Tracers
technology Genetic
Engineering
Synthesis of
Banks of Cloning specific DNA
DNA, RNA Synthesis
probes
and proteins of new Mass prodn. of
proteins human proteins
Complete Localisation of
New types of Resource bank
map of the genetic disorders
plants and for rare human
human
animals chemicals
genome
New
New types antibiotics
of food Gene therapy
What Is Enzyme Immobilization ?

Enzyme immobilization may be defined as a

process of confining the enzyme molecules
to a solid support over which a substrate is
passed and converted to products.

What Is An Immobilized Enzyme?

An immobilized enzyme is one whose

movement in space has been restricted either
completely or to a small limited region.
 Why ImmobilizeEnzymes?
Protection from degradation and deactivation.
Re-use of enzymes for many reaction cycles, lowering the total production
cost of enzyme mediated reactions.

Ability to stop the reaction rapidly by removing the enzyme from the reaction
solution.

Enhanced stability.

Easy separation of the enzyme from the product.

Product is not contaminated with the enzyme.

 An Ideal Carrier Matrices For Enzyme
Immobilization
Inert.
Physically strong and stable.
Cost effective.
Regenerable.
Reduction in product inhibition.
 CLASSIFICATION
CLASSIFICATION OF CARRIERS
OF CARRIERS

Inorganic Organic
Organic
Inorganic Organic
OrganicNatural
Natural
Carriers Synthetic
Synthetic
Carriers Carriers
Carriers
••High
Highpressure
pressure ••Favourable
Carriers
Carriers
Favourable
stability.
stability. compatibility
compatibilitywith
with ••High
Highchemical
chemical
••May
Mayundergo
undergo proteins.
proteins. and
andmechanical
mechanical
abrasion
abrasion Examples:
Examples: stability.
stability.
Examples
Examples:: cellulose derivatives-
1.1.cellulose derivatives- Examples:
Examples:
ooDEAE-cellulose
DEAE-cellulose
Commercialy SiO2
1.1.Commercialy SiO2 ooCM-cellulose.
CM-cellulose.
1.Polystyrene
1.Polystyrene
available
availablematerials-
materials- 2.2.Dextran.
Dextran. 2.Polyvinylacetate
2.Polyvinylacetate
ooPorous
Porousglass.
glass. 3.Polysacharides
3.Polysacharides 3.
3.Acrylic
Acrylic
ooSilica.
Silica. Agarose,
Agarose,Starch
Starch polymers
polymers
2.2.Mineral
Mineralmaterials - -
materials Pectine
Pectine,Chitosan
,Chitosan
(clays)
(clays)
Celite
Celite,Centonite
,Centonite
 METHODS FOR ENZYME
IMMOBILIZATION

PHYSICAL CHEMICAL

ADSORPTION
COVALENT BINDING
ENTRAPMENT

ENCAPSULATION
SUPPORT CROSS
LINKING
 Physical Methods For
Immobilization
ADSORPTION
Involves the physical binding of the enzyme on the surface of carrier matrix.

Carrier may be organic or inorganic.

The process of adsorption involves the weak interactions like Vander Waal
or hydrogen bonds.

Carriers: - silica, bentonite, cellulose, etc.

e.g. catalase & invertase

 ADVANTAGE DISADVANTAGES

1. Simple and economical 1.Relatively low surface

2. Limited loss of activity area for binding.

3.Can be Recycled, 2.Exposure of enzyme to

Regenerated & Reused. microbial attack.

3.Yield are often low due to

inactivation and desorption.
 Entrapment

In entrapment, the enzymes or cells are not directly attached to the support
surface, but simply trapped inside the polymer matrix.

Enzymes are held or entrapped within the suitable gels or fibres.

It is done in such a way as to retain protein while allowing penetration of
substrate. It can be classified into lattice and micro capsule types.
Inclusion in gels: Poly acrylamide gel, Poly vinyl alcohol gels.

Inclusion in fibers: Cellulose and Poly -acryl amide gels.

Inclusion in micro capsules: Polyamine, Polybasic -

acid chloride monomers.

Lattice-Type Entrapment

Entrapment involves entrapping enzymes within the

interstitial spaces of a cross-linked water-insoluble
polymer. Some synthetic polymers such as polyarylamide,
polyvinylalcohol, etc... and natural polymer (starch) have
been used to immobilize enzymes using this technique.
 MicrocapsuleType Entrapmet/
Encapsulation/Membrane Confinement

It involves enclosing the enzymes within semi

-permeable polymer membranes e.g. semi permeable
collodion or nylon membranes in the shape of spheres.
ADVANTAGES DISADVANTAGS

1.No chemical 1. The enzyme may leak

from the pores.
modification.

2.Relatively stable
forms.

3.Easy handling and re-

usage.
 Covalent Binding
Based on the binding of enzymes and water-insoluble carriers by covalent
bonds
The functional groups that may take part in this binding are Amino
group, Carboxyl group, Sulfhydryl group, Hydroxyl group, Imidazole
group, Phenolic group, Thiol group, etc
Disadvantages : covalent binding may alter the conformational structure
and active center of the enzyme, resulting in major loss of activity and/or
changes of the substrate
Advantages : the binding force between enzyme and carrier is so strong
that no leakage of the enzymes occurs, even in the presence of substrate or
solution of high ionic strength.
 Cross Linking
Cross linking involves intermolecular cross linking of enzyme molecules in the
presence/absence of solid support.

The method produces a 3 dimensional cross linked enzyme aggregate

(insoluble in water) by means of a multifunctional reagent that links covalently
to the enzyme molecules.
Advantages of cross linking:-
1. Very little desorption(enzyme strongly bound)
2. Higher stability (i.e. ph, ionic & substrate concentration)

Disadvantages of cross linking:-

1. Cross linking may cause significant changes in the active site.
2. Not cost effective.
Fig. Pictorial representation of different immobilization methods.
Comparison Between The Methods
 Limitations Of Enzyme
Immobilization
Cost of carriers and immobilization.

Changes in properties (selectivity).

Mass transfer limitations.

Problems with cofactor and regeneration.

Problems with multienzymes systems.

Activity loss during immobilization.

BIOSENSOR
What is a Biosensor?
Components of a Biosensor

Detector
 Basic Characteristics of a
Biosensor

1. LINEARITY Linearity of the sensor should be high

forthe detection of high substrate
concentration.
2. SENSITIVITY Value of the electrode response per
substrate concentration.
3. SELECTIVITY Chemicals Interference must be
minimised for obtaining the correct
result.
4.RESPONSE TIME Time necessary for having 95%
of the response.
 Biosensor

1. The Analyte (What do you want to detect)

Molecule - Protein, toxin, peptide, vitamin, sugar,
metal ion

2. Sample handling (How to deliver the analyte to the sensitive region?)

(Micro) fluidics - Concentration increase/decrease),
Filtration/selection
 Biosensor

3. Detection/Recognition
(How do you specifically recognize the analyte?)

4. Signal
(How do you know there was a detection)
 Typical Sensing Techniques
for Biosensors

Fluorescence
DNA Microarray
SPR Surface plasmon resonance
Impedance spectroscopy
SPM (Scanning probe microscopy, AFM,
STM)
QCM (Quartz crystal microbalance)
SERS (Surface Enhanced Raman Spectroscopy)
Electrochemical
Types of Biosensors

1. Calorimetric Biosensor
2. Potentiometric Biosensor
3. Amperometric Biosensor
4. Optical Biosensor
5. Piezo-electric Biosensor
PIEZO-ELECTRIC BIOSENSORS

Piezo-electric devices use gold to detect the

specific angle at which electron waves are
emitted when the substance is exposed to laser
light or crystals, such as quartz, which vibrate
under the influence of an electric field.

.
 ELECTROCHEMICAL BIOSENSORS

• For applied current: Movement of e- in redox

reactions detected when a potential is applied
between two electrodes.
Potentiometric Biosensor

◦For voltage: Change in distribution of charge

is detected using ion-selective electrodes,
such as pH-meters.
 OPTICAL BIOSENSORS

•Colorimetric for color

Measure change in light adsorption

•Photometric for light intensity

Photon output for a luminescent or

fluorescent process can be detected with
photomultiplier tubes or photodiode
systems.
 CALORIMETRIC BIOSENSORS

If the enzyme catalyzed reaction is exothermic,

two thermistors may be used to
measure the difference in resistance
between reactant and product and, hence,
the analyte concentration.
Electrochemical DNA Biosensor
 Steps involved in electrochemical
DNA hybridization biosensors:
 Formation of the DNA recognition layer

 Actual hybridization event

 Transformation of the hybridization event

into an electrical signal
 DNA biosensor

Motivated by the application to clinical diagnosis

and genome mutation detection

Types DNA Biosensors

Electrodes
Chips
Crystals
 Application of Biosensor

 Food Analysis
 Study of biomolecules and their interaction
 Drug Development
 Crime detection
 Medical diagnosis (both clinical and laboratory use)
 Environmental field monitoring
 Quality control
 Industrial Process Control
 Detection systems for biological warfare agents
 Manufacturing of pharmaceuticals and replacement
organs
 47

GENETIC
ENGINEERING
 48

Genes are the fundamental basis of all life,

determine the properties of all living forms of
life, and are defined segments of DNA.
Because DNA structure and composition in all
living forms is essentially the same, any
technology that can isolate, change or
reproduce a gene is likely to have an impact on
almost every aspect of society.
 49

Genetic engineering has been defined as the

formation of new combinations of heritable
material by the insertion of nucleic acid
molecules, into any virus, bacterial plasmid or
other vector system so as to allow their
incorporation into a host organism in which
they do not naturally occur, but in which they
are capable of continued propagation.
50

1
 51

gene technology is the modification of the

genetic properties of an organism by the use of
recombinant DNA technology.
Genes may be viewed as the biological
software and are the programs that drive the
growth, development and functioning of an
organism.
By changing the software in a precise and
controlled manner, it becomes possible to
produce desired changes in the characteristics
of the organism.
 52

These techniques allow the splicing of DNA

molecules of quite diverse origin, and, when
combined with techniques of genetic
transformation etc., facilitate the introduction of
foreign DNA into other organisms.
The foreign DNA or gene construct is introduced
into the genome of the recipient organism host
in such a way that the total genome of the host
is unchanged except for the manipulated
gene(s).
 53

While traditional plant and animal genetic breeding

techniques also change the genetic code it is
achieved in a less direct and controlled manner.
Genetic engineering will now enable the breeder to
select the particular gene required for a desired
characteristic and modify only that gene.
Although much work to date has involved bacteria,
the techniques are evolving at an astonishing rate
and ways have been developed for introducing
DNA into other organisms such as yeasts and
plant and animal cell cultures.
 54

Life forms containing ‘foreign’ DNA are termed

transgenic
These methods potentially allow totally new
functions to be added to the capabilities of
organisms, and open up vistas for the genetic
engineering of industrial microorganisms and
agricultural plants and animals that are quite
breathtaking in their scope.
This is undoubtedly the most significant new
technology in modern bioscience and
biotechnology.
 55

In industrial microbiology it will permit the

production in microorganisms of a wide range
of hitherto unachievable products such as
human and animal proteins and enzymes such
as insulin and chymosin (rennet)
in medicine, better vaccines, hormones and
improved therapy of diseases;
in agriculture, improved plants and animals for
productivity, quality of products, disease
resistance, etc;
 56

in food production, improved quality, flavour,

taste and safety;
in environmental aspects, a wide range of benefits
such as pollution control can be expected.
In microbial technology these techniques will be
widely used to improve existing microbial
processes by improving stability of existing
cultures and eliminating unwanted side products.
However, there are many who view genetic
engineering as a transgression of normal life
processes that goes well beyond normal evolution.
 57

Genetic engineering holds the potential to extend the range and

power of almost every aspect of biotechnology.
It is confidently anticipated that within this decade recombinant DNA
techniques will form the basis of new strains of microorganisms
with new and unusual metabolic properties.
In this way fermentations based on these technical advances could
become competitive with petrochemicals for producing a whole
range of chemical compounds, for example ethylene glycol (used
in the plastics industry) as well as improved biofuel production.
In the food industry, improved strains of bacteria and fungi are now
influencing such traditional processes as baking and cheese-
making and bringing greater control and reproducibility of flavour
and texture.
 58

A full understanding of the working concepts of

recombinant DNA technology requires a good
knowledge of molecular biology.
The basic molecular techniques for the in vitro
transfer and expression of foreign DNA in a
host cell (gene transfer technology), including
isolating, cutting and joining molecules of DNA,
and inserting into a vector (carrying) molecule
that can be stably retained in the host cell,
were first developed in the early 1970s.
 59

2
 60

Cutting DNA molecules:

DNA can be cut using mechanical or enzymatic
methods.
The non-specific mechanical shearing will
generate random DNA fragments
In contrast, when specific restriction
endonuclease enzymes are used it is possible
to recognise and cleave specific target base
sequences in double-stranded (ds) DNA.
 61

Large numbers of different restriction

endonucleases have been extracted and
classified from a wide variety of microbial
species.
Restriction endonucleases are named according
to the species from which they were first
isolated, e.g. enzymes isolated from
Haemophilus influenzae strain Rd are
designated Hind and when several different
restriction enzymes areisolated from the same
organism they are designated HindI, HindII etc.
 62

Splicing DNA:
DNA fragments can be joined together in vitro
by the action of specific DNA ligases.
The DNA ligase that is widely used was
encoded by phage T4.
The composite molecules in which DNA has
been inserted have also been termed ‘DNA
chimaeras’
 63

The vector or carrier system:

Two broad categories of expression vector
molecules have been developed as vehicles for
gene transfer, plasmids (small units of DNA distinct
from chromosomes) and bacteriophages (or
bacterial viruses).
Vector molecules should be capable of entering
the host cell and replicating within it.
Ideally, the vector should be small, easily prepared
and must contain at least one site where
integration of foreign DNA will not destroy an
essential function.
 64

Introduction of vector DNA recombinants:

The new recombinant DNA can now be
introduced into the host cell and if acceptable
the new DNA will be cloned with the
propagation of the host cell.
Novel methods of ensuring DNA uptake into
cells include electroporation and mechanical
particle delivery or biolistics.
 65

Electroporation is a process of creating transient pores in the

cell membrane by application of a pulsed electric field.
Creation of such pores in a membrane allows introduction of
foreign molecules, such as DNA, RNA, antibodies, drugs,
etc., into the cell cytoplasm.
Development of this technology has arisen from synergy of
biophysics, bioengineering and cell and molecular biology.
While the technique is now widely used to create transgenic
microorganisms, plants and animals, it is also being
increasingly used for application of therapeutics and gene
therapy.
 66

The mechanical particle delivery or ‘gene gun’

methods deliver DNA on microscopic particles
into target tissue or cells. This process is
increasingly used to introduce new genes into a
range of bacterial, fungal, plant and
mammalian species and has become a main
method of choice for genetic engineering of
many plant species including rice, corn, wheat,
cotton and soybean.
 67

3
 68

4
B’ PHARMACY
SUBJECT: Pharmaceutical Biotechnology
Subject Code: BP605T
MODULE- 2

GENE CLONING VECTORS

PRESENTED BY Chamanpreet Kaur

Assistant Professor
Pharmaceutics
ASBASJSMCOP , BELA
 Objective of course
Genetic Engineering applications relation to production of pharmaceutical

• Learning Outcome

• Students will learn about cloning vectors. They will also learn
about the application of genetic Enginnering and various steps
of Polymerase Chain Reaction.
 INTRODUCTION

• A cloning vector is a DNA molecule in which

foreign DNA can be inserted or integrated and
which is further capable of replicating within
host cell to produce multiple clones of
recombinant DNA.

• Examples: Plasmids,phage or virus

 Characteristics

It should be able to replicate autonomously.

Origin of replication.
Selectable markers.
Restriction sites.
 Types

• Plasmid as vector .

• Bacteriophage as vector.

• Cosmid as vector

• Phagemid as vector.
 P lasmid Vector: pB R 3 2 2

• Contains:
• Selectable Markers:
•Ampicillin resistance gene.
•Tetracycline resistance gene.
4362 bp
• Col E I replication origin. Eco RI site.

Structure of E.Coli plasmid cloning vector pBR322

Screening procedure of cloning vector

Blue/White selection.

replica plating technique.

 B lue/ W hite Selection
Bacteria with
plasmid plus insert

IPTG + X-Gal

Overnight growth

Only colonies
Colonies with insert - white
from bacteria that
Colonies w/o insert - blue
have plasmid
Replica Plating Technique
 BACTERIOPHAGE VECTORS
• It infects bacteria.
• Follow either lytic or lysogenic cycle.
• Lambda phage vector
• high transformation efficiency, about 1000
times more efficient than the plasmid vector.
• Origin of replication.
• genes for head and tail protein.
• single- stranded protruding cohesive ends.
• Size is 48,502 bp.
 Cosmid vector
• Combine parts of the lambda chromosome
with parts of plasmids.
• an origin of replication (ori).
• a cos site(a sequence yield cohesive end) .
• an ampicillin resistance gene (amp),
• restriction sites for cloning
• Cosmids can carry up to 50 kb of inserted
DNA.
 APPLICATION
• A particular gene can be isolated and its
nucleotide sequence determined
• Control sequences of DNA can be identified&
analyzed
• Protein/enzyme/RNA function can be
investigated
• Mutations can be identified, e.g. gene defects
related to specificdiseases
• Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production.
PRODUCTION OF
HEPATITIS
PRODUCTION OF INSULIN
Polymerase Chain Reaction (PCR)
 DNA
DNA is a nucleic acid that is composed of
two complementary nucleotide building
block chains.

The nucleotides are made up of a phosphate

group, a five carbon sugar, and a nitrogen
base.
 DNA
 DNA Sugar
– Deoxyribonucleic acid

 RNA Sugar
– Ribonucleic acid
 DNA
DNA has four nitrogen bases.

 Two are purines ( 2 ringed base )

– Adenine ( A ), Guanine ( G )

 Two are pyrimidines ( 1 ringed base )

– Cytosine ( C ), Thymine ( T )
 DNA
These four bases are linked in a repeated
pattern by hydrogen bonding between the
nitrogen bases.

The linking of the two complementary

strands is called hybridization.
 DNA
Example of bonding pattern.

Primary strand
CCGAATGGGATGC
GGCTTACCCTACG
Complementary strand
 DNA
A purine always links with a pyrimidine base
to maintain the structure of DNA.

Adenine ( A ) binds to Thymine ( T ), with

two hydrogen bonds between them.

Guanine ( G ) binds to Cytosine ( C ), with

three hydrogen bonds between them.
 WHY WE NEED PCR?
• Template (the DNA you are exploring)
• Sequence-specific primers flanking the target
sequence, Forward & Reverse.
• Polymerases
• Nucleotides (dATP, dCTP, dGTP, dTTP)
• Magnesium chloride (enzyme cofactor)
• Buffer
• Water, mineral oil
 PCR REQUIREMENTS
 Magnesium chloride: .5-2.5mM
 Buffer: pH 8.3-8.8
 dNTPs: 20-200µM
 Primers: 0.1-0.5µM
 DNA Polymerase: 1-2.5 units
 Target DNA:  1 µg
• Denaturation 93 to 95°C 1min

• Annealing 50 to 55°C 45 sec.

• Elongation 70-75°C 1-2 min.

 PCR CYCLE REVIEW
 Denaturation: 94°- 95°C
 Primer Annealing: 55°- 65°C
 Elongation of DNA: 72°
 Number of Cycles: 25-40
 No target products are made until the
third cycle.
 At 30 cycles there are 1,073,741,764
target copies (~1×109).
 ADVANTAGES OF PCR
 Speed
 Ease of use
 Sensitivity
 Robustness
 APPLICATION OF PCR
 Screening human DNA samples for
mutations associated with genetic
diseases such as thalassemia and cystic
fibrosis.
 Can detect the presence of viral DNA before it
turns in to a killer.
 PCR enables rapid amplification of template DNA
for screening of uncharacterized mutations
 Can obtain sequences from hair, blood stain,
bones, other forensic specimens, other remains
preserved at archaeological sites.
B Pharmacy
Subject- Pharmaceutical Biotechnology
Sub Code- BP605T

MODULE- 3

Chamanpreet Kaur
Assistant Professor
ASBASJSM COLLEGE OF PHARMACY, BELA

1
Objective of Course
Understanding the importance of enzymes in Pharmaceutical
Industries.

Learning Outcomes
1) Students will learn about various types of immunity including
Immunoglobulins, MHC and hypersensitivity reactions.
2) They will also learn about hybridoma technology,
immunostimulation and immunosuppressions.
3) They will also able to know about stoarage and stability conditions
of official vaccines.
 Cellular Immunity
It is an adapative immune response that is principally
delibrated by thymus- derived small lymphocytes,
which are recognized as T cells. They do not terminate
infected cells or pathogens, however they trigger and
direct further immune cells to do so. Cellular immunity
is a protective immune process that involves the
activation of phagocytes, antigen-sensitized cytotoxic
T cells and the release of cytokines and chemokines in
response to antigen. Cellular immunity is most
effective against cells infected with viruses,
intracellular bacteria, fungi and protozoans, and
cancerous cells. It also mediates transplant rejection.
 Humoral Immunity
• Results in production of
proteins called
“immunoglobulin's” or
“antibodies”.
• Body exposed to
“foreign” material
termed “antigen” which
may be harmful to
body:
virus, bacteria, etc.
• Antigen has bypassed other
protective
mechanisms, ie, first and
second line of defense. 2
CLASSES (ISOTYPES) OF IMMUNOGLOBULINS

• Classes based on constant region of heavy chains

– Immunoglobulin A (IgA)
– Immunoglobulin D (IgD)
– Immunoglobulin E (IgE)
– Immunoglobulin G (IgG)
– Immunoglobulin M (IgM)
• Differentiation of heavy chains
– Length of C region, location of disulfide bonds, hinge
region, distribution of carbohydrate
• Classes have different effector functions

5
 Immunoglobulin Classes
I. IgG
 Structure: Monomer
 Percentage serum antibodies: 80%
 Location: Blood, lymph, intestine
 Half-life in serum: 23 days
 Complement Fixation: Yes
 Placental Transfer: Yes
Known Functions: Enhances
phagocytosis, neutralizes toxins and
viruses, protects fetus and newborn.
6
 Immunoglobulin Classes
II. IgM
 Structure: Pentamer
 Percentage serum antibodies: 5-10%
 Location: Blood, lymph, B cell surface (monomer)
 Half-life in serum: 5 days
 Complement Fixation: Yes
 Placental Transfer: No
Known Functions: First antibodies produced
during an infection. Effective against microbes
and agglutinating antigens.
7
 Immunoglobulin Classes
III. IgA
 Structure: Dimer
 Percentage serum antibodies: 10-15%
 Location: Secretions
(tears, saliva, intestine, milk), blood and lymph.
 Half-life in serum: 6 days
 Complement Fixation: No
 Placental Transfer: No
Known Functions: Localized protection of
mucosal surfaces. Provides immunity to infant
digestive tract. 8
 Immunoglobulin Classes
IV. IgD
 Structure: Monomer
 Percentage serum antibodies: 0.2%
 Location: B-cell surface, blood, and lymph
 Half-life in serum: 3 days
 Complement Fixation: No
 Placental Transfer: No
Known Functions: In serum function is unknown.
On B cell surface, initiate immune response.

9
 Immunoglobulin Classes
V. IgE
 Structure: Monomer
 Percentage serum antibodies: 0.002%
Location: Bound to mast cells and basophils
throughout body. Blood.
 Half-life in serum: 2 days
 Complement Fixation: No
 Placental Transfer: No
Known Functions: Allergic reactions. Possibly
lysis of worms.
10
 CLASSES (ISOTYPES) OF
IMMUNOGLOBULINS
• Additional classification
based on light chains
– Kappa
– Lambda
• Each IG has either kappa or
lambda, not both
– IgG kappa
– IgG lambda
• No functional differences
between light chains

11
 IgE AND IgD ANTIBODIES OF THE
IMMUNE RESPONSE
• IgE
– Binds with high affinity to receptors on mast
cells, basophils and activated Eosinophi ls
– Longer half-life when cell bound
– Initiates a strong inflammatory reaction to parasites
– Involved in allergic reactions
• IgD
– Antigen receptor on mature B-cells
– No other known function
12
MHC (Major Histocompatibility
Complex)
It is defined as set of genes that code for cell surface proteins essential for the
acquired immune system to recognize foreign molecules in vertebrates, which
in turn determines histocompatiblity. It is of four types.
1) Class 1 molecules
2) Class 2 molecules
3) Class 3 molecules

FUNCTIONS OF MHC
1) It binds to endogenous antigen and present to T helper cells.
2) They are found on surface of all nucleated cells.
3) They are secreyed protein possesing immune funmctions.
4) They are also involved in complement activation.
5) They involved in inflammation caused by cytokines and tumor
necrosis factor.
 Hypersensitivity

• Hypersensitivity refers to having an extreme

sensitivity to stimulation of the senses, i.e., touch,
sight, hearing, taste, and smell. Children who are
hypersensitive may complain about sensory
stimuli that seem minor to others. They also find
it almost impossible to regulate their emotional
and behavioral responses to high levels of sensory
stimulation. For example, a hypersensitive child
may lose his temper as a result of being hugged.
 Causes of Hypersensitivity
• There are several known causes of hypersensitivity in children, including:
• Sensory processing disorders, including sensory integration disorder
• Pervasive developmental disorders, including autism
• Neurological disorders, including peripheral neuropathy
• Traumatic brain injury
• Fibromyalgia
• Structural abnormalities in any one of the sensory systems, e.g., having a deviated
septum
• Damage to any one of the sensory systems, e.g., damage to your auditory system
caused by overexposure to loud noises
• In addition, the following disorders have been linked to hypersensitivity in children:
• Anxiety disorders, such as obsessive-compulsive disorder
• ADHD
• Fragile X syndrome
• Down syndrome
• Cerebral palsy
• Epilepsy
 Types of Hypersensitivity
• There are five types of hypersensitivity in
children. Each type of hypersensitivity is very
uncomfortable to the child that experiences it.
They are:
• Hypersensitivity to touch and movement
• Visual hypersensitivity
• Auditory hypersensitivity
• Hypersensitivity to taste
• Hypersensitivity to smell
 Immune suppression
It is a reduction of the activation or efficacy of the immune system. It is a
situation in which the body’s immune system is intentionally stopped from
working or is made less effective, usually by drugs, especially in order to
help the body to accept an organ that has been taken from another person’s
body.
It is of two types:
1) Deliberately induced immunosuppression: It occurs in case of medications
and during any organ transplantations.
2) Non- Deliberate immunosuppression: It occurs in case of many
complement deficiencies like many types of cancer, chronic infection and
human immunodeficiency virus (HIV).
 GENERAL METHODS USED FOR PREPARATION OF VACCINES

Cholera Vaccine ( Bacterial vaccine)

Cholera is serious infection which is caused by spirillium vibrio cholera,
cholera vaccine is homogenous suspension which is prepared from killed
cholera vibrio’s strain having antigenic efficacy and purity
Category: Active immunizing agent
Dose: Initial dose is 0.5ml and second dose is 1.0 ml
Storage: Store at temp between 2-8°C
BCG Vaccine
BCG is a suspension of living cells, which is obtained from
Mycobacterium tuberculosis which is known as bacillus calmette
guerin.BCG vaccine is a live freeze dried vaccine which is used for
prevention of tuberculosis.
It is a freeze dried prepartion stored in dark 2-8°C.
Should be protected from light.
Its single dose 0.1ml.
Diptheria Vaccine
Diptheria is a communicable disease which is caused by Corneybacterium
diptheria which colonizes and forms a pseudo membrane at the infection
site. Diptheria toxin is synthesized and secreted as a single polypeptide
chain which is having A Chain and B Chain.
It is an acti9ve immunizing agent.
Its initial dose is 0.5ml, second dose is 0.5ml after 4 weeks and third dose
is 0.5ml after another 4 weeks.
It should not contaion more than 30LF of diptheria toxoid per dose of
0.5ml.
Polio Vaccine ( viral vaccine)
Polio is a live oral aqueous suspension. It is obtained from attenuated
strains of polio myelitis virus. There are three distinct antigenic types of
polio myelitis types 1,2,3 and each has much greater antigenic stability.It
should be stored in sealed tight resistant containers at a temprature between
2-8°C .
It has potency not les than 2.5 units for each dose.
It is freeze dried powder or pellets which in reconstituted with suitable
solvent.
Tetanus (Antitoxin)
Tetanus antotoxin is a liquid preparation which is containing specific anto-
toxin globulins which are obtained by hyper immune sysytem of horse and
other suitable animals. These liquid preparations contains suitable
antimicrobial preservative.
It is passive immunizing agent.
It should be givenm by subcutaneous and intramuscular injection.
Tetanus toxoid should be stored between 2-8°C.
It is freeze dried or pale yellow liquid or cream coloured powder and
pellets.
 HYBRIDOMATECHNOLOGY
• Hybridoma technology is a method for producing large number of
identical antibodies called monoclonal antibodies.
• It was discovered by G.kohler and C.milstein in 1975. they were
awarded nobel prize for physiology and medicine in 1975.
• The hybrid cells are produced by fusing B- lumphocyte with
myeloma cells or tumour cells.
• The B-lymphocyte have the ability to produce large number of
antibodies and tumour cells have indefinite growth.
• This is why two cells are used for the production of hybrid cell.
Procedure
1. The mouse is immunised by specific antigen injection against which
monoclonal antibodies have to be produced.
2. After 72 hrs of immunisation spleen is collected from
mouse.(antibody prodcing B cells).
3. The B cells are fused with immortalised myeloma cells by
polyethylglycol or sendai virus.
4. The B cells are fused with immortalised myeloma cells.
5. The fused cells are incubated in the HAT medium.
 HAT medium
• The hybridoma cells or fused cells are selected
using selective media are called HAT medium.
• It contains Hypoxanthine, Aminopterin and
Thymidine.
• The unfused B cells will die due to their short life
span.
• The myeloma cells can synthesise DNA nucleotides
using two pathway : Denovo pathway and salvage
pathway.
• In HAT medium, the myeloma cells are unable to replicate because the denovo
pathway is blocked by aminopterin in the medium.
• When denovo pathway is blocked, the cell will utilise salvage as an
alternative pathway. But it cannot takesplace due to the lack of HGPRT
(Hypoxanthine-guanine phosphoribosyl transferase).
• so it is contributed by B cell and is rich in HGPRT.
• The salvage pathway is also inhibited due to the mutation of Thymidine
Kinase (TK), an enzyme that catalyses the phosphorylation reaction.
• The resulting clones of hybridoma cells secrets large quantities of
monoclonal antibodies.
 Applications
• It is used for the early detection of pregnancy.
• Detection and treatment of cancer.
• Diagnosis of leprosy.
• Treatment of autoimmune diseases.
• Radiolabelled monoclonal antibodies are used in vivo for detecting or
locating tumour antigen.
• Used for making immunotoxins that inhibit protein synthesis.
• Eg: ricin, shigella toxin & diphteria toxin.