Biotechnology

Biotechnology
Biotechnology is the application of scientific and engineering principles

to the process of materials by biological agents to provide better goods
and services.
In another word, Biotechnology is the application of biological
organisms, systems and processes for manufacturing and service
industry.
Biotechnology refers to the use of microorganisms, such as bacteria or
yeasts, or biological substances, such as enzymes, to perform specific
industrial or manufacturing processes. Applications include the
production of certain drugs, synthetic hormones, and bulk foodstuffs as
well as the bioconversion of organic waste and the use of genetically
altered bacteria in the cleanup of oil spills.

The United Nations Convention on Biological Diversity defines
biotechnology as: "Any technological application that uses biological
systems, living organisms, or derivatives thereof, to make or modify
products or processes for specific use."

Sub-fields of biotechnology:
There are number of jargon terms for sub-fields of biotechnology.
Red biotechnology is biotechnology applied to medical processes.
An example would include an organism designed to produce an
antibiotic, or engineering genetic cures to diseases through genomic
manipulation.
White biotechnology, also known as grey biotechnology, is
biotechnology applied to industrial processes. An example would
include an organism designed to produce a useful chemical. White
biotechnology tends to consume less resources that traditional process
when used to produce industrial goods.

Green biotechnology is biotechnology applied to agricultural
processes. An example would include an organism designed to grow
under specific environmental conditions or in the presence (or
absence) of certain agricultural chemicals. Green biotechnology tends
to produce more environmentally friendly solutions than traditional
industrial agriculture. An example of this would include a plant
shahid.islam22@yahoo.com
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engineered to express a pesticide, thereby eliminating the need for
external application of pesticides.

The term blue biotechnology has also been used to describe the
marine and aquatic applications of biotechnology, but its use is
relatively rare.
What is the difference between biotechnology and

pharmaceutical biotechnology?
Biotechnology is the study of cell and other living organisms for the
development of novel drugs and therapies whereas pharmaceuticals is
study of in organic chemical combinations [do not have life] for the
development of drugs or therapies.

Pharmaceutical biotechnology is (might be) the study of micro- and
macro-organisms and hybridomas to create pharmaceuticals that are
safer and more cost-effective than conventionally produced
pharmaceuticals.
Difference between genetic engineering and biotechnology?
Genetic engineering is the part of biotechnology. It deals with altering
the original function of gene and inserting the desired one. For example;
inserting the human insulin gene in E.coli.
Subjects of Biotechnology:
I. Microbiology
II. Cell biology
III. Biochemistry
IV. Molecular biology
V. Genetics
VI. Engineering technology
VII. Biophysics
Branches of Biotechnology:
1. Agricultural biotechnology
2. Medical Biotechnology: It is one of the areas of medical sciences which
utilize the Diagnostic aids like the AIDS detection kits, glucose
measuring kits etc, and Attempts for correction of some hereditary
disorders by gene incorporation utilizing the Basic concepts of
biotechnology. The production of artificial organs like the liver and
kidney are the emerging field of the subject
3. Engineering biotechnology
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4.
5.
6.
7.
8.
Biomedical engineering:
Textile and paper biotechnology
Environmental biotechnology
Leather biotechnology
Pharmaceutical Biotechnology.so on.
Pharmaceutical Biotechnology:
A field that uses micro- and macro-organisms and hybridomas to
create pharmaceuticals that are safer and more cost-effective than
conventionally produced pharmaceuticals. This is major branch of
biotechnology which are involved in the production of
The therapeutic proteins and hormones, fermentation products like the

antibiotics, specially designed vaccines or drug design using the
receptor hypothesis, gene Correction, drug delivery to specific tissues,
production control using Biosensors, standardization of
chemotherapeutic agents and the diagnostic aids by Employing a
number of techniques such as gene cloning technology, recombinant
DNA Technology, enzyme immobilization, fermentation technology,
hybridoma technology, Mutagenesis etc.

History of biotechnology:
1953: DNA structure proposed by Watson and Crick, and Franklin.
1960: Arthur Kornberg synthesizes DNA in vitro.
1970: Cetus founded at Berkeley.
1970: Hamilton Smith and Kent Wilcox isolate the first restriction
enzyme.
1972: Paul Berg uses a restriction enzyme to form a hybrid circular
molecule.
1973: Stanley Cohen and Herbert Boyer develop DNA cloning and
recombinant DNA.
1975: Asilomar conference discussing the ethics of recombinant DNA
research. First monoclonal antibodies produced.
1976: Robert Swanson and Herbert Boyer found Genentech
Guidelines from the NIH prohibit some categories of recombinant
DNA experiments.DNA sequencing introduced.
1977: Genentech clones hormone somatostatin in bacteria, the first
cloning of a protein using a synthetic recombinant gene.
1978: Biogen founded by Gilbert and Sharp. Genentech announces
successful.
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1979: NIH guidelines relaxed. Genentech clones human growth

hormone.
1980: Amgen founded. Supreme Court rules living organisms could
be patented production of human insulin. Bacterial DNA introduced
into yeast chromosome.
The 1980s were a boom for industrial microbiology. Pharmaceutical
companies developed the strategy of drugs from genetically
engineered organisms.
1980: Leroy Hood and Mike Hunkapiller develop protein sequencer
Fred Sanger develops shotgun method for sequencing genomes.
1981: Applied Bio systems founded by Hood. During the 80s they
invented the automated protein synthesizer, protein sequencer, DNA
synthesizer, and DNA sequencer.
1983: Kary Mullis invents PCR.
1983: HIV genome cloned and sequenced by Chiron.
1986: EPA approves release of genetically altered tobacco, first
engineered crop.
A-interferon and first recombinant vaccine
for hepatitis B was approved.

1988: Leder and Stewart awarded patent for mouse breast-cancer
model, First patent for genetically altered animal.

Major corporate players were: Genentech, Biogen, Cetus, and
Genex.
Insulin, -interferon, hepatitis B vaccine, interleukin-2
Biotechnology Applications for world food production

According to FAO: Plant tissue culture can help developing countries produce diseasefree, high quality planting material. In commercial applications, such as
floriculture, it also generates much-needed employment, particularly
for women.
DNA-based techniques include isolation, amplification, modification
and recombination of DNA; genetic engineering to obtain genetically
Modified Organisms (GMOs); use of markers and probes in gene
mapping and in functional and structural genomics; and unambiguous
identification of genotypes through DNA fingerprinting.

Diagnostic kits based on the products of biotechnology (monoclonal
antibodies, recombinant antigens) are very important modern
agricultural applications for identification of plant and animal
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pathogens, with economic implications for pathogen monitoring and
control programmes.
Agro-industrial applications - There is untapped potential for
increasing employment and adding value to agricultural products
through agroindustry, diversification and alternative utilization of raw
materials (e.g., use of vegetable oils as biofuels)
Biotechnology: Biotechnology is the application of recombinant DNA
techniques in commercial enterprises. There are three main areas:

1) Industry;
2) Agriculture;
3) Medicine/Pharmaceuticals.
1) Industry: The following are examples of the diverse use of biotechnology
in industry:
Alternative fuels -- the production of ethanol from the digestion of
biomass by genetically improved yeast or bacteria.

Enzyme production -- genetically modified bacteria produce
enzymes for use in research and industry. Example: bacterial protease
used in laundry detergent.

Biodegradable plastics -- polyhydroxybutyrate (PHB) and
polyhydroxyalkanoate
(PHA) are found naturally in some bacteria. Through gene transfer,
plants that produce PHB in inclusion bodies have been made.
2) Agriculture: Extensive use of biotechnology has been made to modify
both plants and animals used in agriculture. This frequently involves the
development of transgenic plants and animals through the insertion of
genes from other species into their genomes. Improvements to
commercial crops include: disease, drought, insect, and pesticide
resistance; improved nutrient content, extended fruit shelf life, and
pharmaceutical delivery.
3) Medicine/Pharmaceuticals:
At present, up to 70% of the commercial biotechnology industry is
involved in the production of pharmaceuticals and medically-related
products. A few of the many applications are listed below, followed by
more detailed descriptions of selected areas:
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Gene therapy -- the direct use of DNA or RNA to treat disease, usually
by the introduction of a functional gene into a cell to correct a disease
state.
Foreign protein expression systems -- examples: yeast that
express Hepatitis B antigens for use in vaccine and bacteria that
express human insulin.

Stem cell research -- uses the manipulation of undifferentiated cells
to replace damaged or diseased tissues in the body.

Cancer genetics -- looking for ways to selectively inhibit genes
responsible for cancer formation, growth, and treatment.
Genetic engineering:
Genetic engineering, also called genetic modification, is the human
manipulation of organisms genetic material in a way that does not
occur under natural conditions. It involves the use of recombinant
DNA techniques, but does not include traditional animal and plant
breeding or mutagenesis.
Any organism that is generated using these techniques is
considered to be a genetically modified organism. The first
organisms genetically engineered were bacteria in 1973 and then mice
in 1974. Insulin producing bacteria were commercialized in 1982 and
genetically modified food has been sold since 1994.

Genetic engineering, genetic modification (GM), and gene
splicing (once in widespread use but now deprecated) are terms for
the process of manipulating genes in an organism, usually outside of
the organism's normal reproductive process.
It often involves the isolation, manipulation and reintroduction of
DNA into model organisms, usually to express a protein. The aim
is to introduce new characteristics to an organism to increase its
usefulness such as, increasing the yield of a crop species, introducing a
novel characteristic, or producing a new protein or enzyme. Examples
are the production of human insulin through the use of modified
bacteria and the production of new types of experimental mice like the
OncoMouse, (cancer mouse) for research, through genetic redesign.
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In short, genetic engineering is the technology of preparing

recombinant DNA in vitro by cutting up DNA molecules and splicing
together fragments from more than one organism.

Recombinant DNA
Recombinant DNA refers to a collection of techniques for creating
(and analyzing) DNA molecules that contain DNA from two unrelated
organisms. One of the DNA molecules is typically a bacterial or viral DNA
that is capable of accepting another DNA molecule; this is called a vector
DNA. The other DNA molecule is from an organism of interest, which could
be anything from a bacterium to a whale, or a human. Combining these
two DNA molecules allows for the replication of many copies of a specific
DNA. These copies of DNA can be studied in detail, used to produce
valuable proteins, or used for gene therapy or other applications.
Recombining of DNA Technology

Recombining of DNA molecules from two different species that are
inserted into a host organism to produce new genetic combinations that
are of value to science, medicine, agriculture, or industry.

Applications of Recombining DNA Technology:
1. Using this technology, scientists are able to isolate a gene, determine
its nucleotide sequence, study its transcripts, mutate it in highly
specific ways, and reinsert the modified sequence into a living
organism.
2. The processes of DNA cloning and sequencing are used to compare
different organisms for evolutionary relatedness and to determine
gene function.
3. Recombinant DNA technology can also be used to study mutations
and their biological effects, such as the role of specific mutations in
disease or abnormal drug response.
4. Other applications of recombinant DNA technology include gene
therapy, reverse genetics, diagnostics, genomics, and protein
manufacture (the preparation of large amounts of protein for basic
research or medicinal use, such as commercially produced insulin).

Process of Recombining DNA Technology:
Producing genetically modified organisms is a multi-step process. It
first involves the isolating and copying the genetic material of interest. A
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construct is built containing all the genetic elements for correct

expression. This construct is then inserted into the host organism, either
by using a vector or directly through injection, in a process called
transformation. Successfully transformed organisms are then grown
and the presence of the new genetic material is tested for.

Examples of Recombining DNA Technology:
Genetic engineering techniques have been applied to various
industries, with some success. Medicines such as insulin and human
growth hormone are now produced in bacteria, experimental mice such as
the oncomouse and the knockout mouse are being used for research
purposes and insect resistant and/or herbicide tolerant crops have been
commercialized. Plants that contain drugs and vaccines, animals with
beneficial proteins in their milk and stress tolerant crops are currently
being developed.
Essential steps involved in the expression of protein genes:
Gene expression
"Gene expression" means the production of a protein or a functional
RNA from its gene. Several steps are required:
Transcription: A DNA strand is used as a template to synthesize a
complementary RNA strand, which is called the primary transcript.

RNA processing: This step involves modifications of the primary
transcript to generate a mature mRNA (for protein genes) or a
functional tRNA or rRNA. For RNA genes (tRNA and rRNA), the
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expression is complete after a functional tRNA or rRNA is generated.
However, protein genes require additional steps:

Nuclear transport: mRNA has to be transported from the nucleus to the
cytoplasm for protein synthesis.

Protein synthesis: In the cytoplasm, mRNA binds to ribosomes, which
can synthesize a polypeptide based on the sequence of mRNA.
The central dogma:

According to the above process, the flow of genetic information is in the
following direction:
DNA > RNA > Protein.
This rule was dubbed the "central dogma", because it was thought that
the same principle would apply to all organisms. However, we now know
that for RNA viruses, the flow of genetic information starts from RNA.
Major techniques and technologies currently used in rDNA
technology:
1. Restriction endonucleases: Cutting of DNA.
2. DNA ligases: Joining of DNA.
3. Reverse transcriptase: Conversion of mRNA to cDNA.
4. Chemical DNA synthesis: Oligonucleotides.
5. Polymerase chain reaction (PCR): Rapid amplification of specific DNA
Segment.
6. Vectors for gene isolation and expression.
7. DNA sequencing techniques.
8. Protein overproduction: Appropriate host and vector system.
Steps involved in the preparation of recombinant DNA:

1. Selection of DNA of interest (foreign DNA).
2. A cloning vector or vehicle to carry inserted pieces of target DNA.
3. Restriction endonucleases which makes internal cuts at specific sites
on DNA.
4. DNA ligase: The enzyme that joins pieces of nucleotides/DNA together.
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5. A host to replicate the vector containing foreign DNA. This may be
prokaryotic
or eukaryotic cell.
6. Screening test for recombinants produced by insertion of DNA.
DNA Cloning:
DNA cloning is a technique to reproduce DNA fragments. It can be
achieved by two different approaches: (1) cell based, and (2) using
polymerase chain reaction (PCR). In the cell-based approach, a vector
is required to carry the DNA fragment of interest into the host cell. The
following figure shows a typical procedure by using plasmids as the
cloning vector.
Cloning Vectors:
"Vector" is an agent that can carry a DNA fragment into a host cell.
If it is used for reproducing the DNA fragment, it is called a "cloning
vector". If it is used for expressing certain gene in the DNA fragment, it
is called an "expression vector".
Commonly used vectors include plasmid, Lambda phage, cosmid and
yeast artificial chromosome (YAC).

Plasmid:
Plasmids are circular, double-stranded DNA molecules that exist in
bacteria and in the nuclei of some eukaryotic cells. They can replicate
independently of the host cell. The size of plasmids ranges from a few kb
to near 100 kb.
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Restriction enzymes:
The procedure used in artificial DNA recombination is similar to the
natural transpositional recombination. The major difference is that
researchers can choose any appropriate enzymes to cut the DNA
molecules. They are usually isolated from bacteria. The role of these
enzymes in bacteria is to "restrict" the invasion of foreign DNA by cutting
it into pieces. Hence, these enzymes are known as restriction enzymes.
Table: Commonly used restriction enzymes:
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Isolation of recombinants DNA clones:
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Figure: The essential steps in DNA cloning using plasmids as

vectors.
a) DNA recombination. The DNA fragment to be cloned is inserted into a

vector The recombinant vector must also contain an antibioticresistance gene.
b) Transformation. The recombinant DNA enters into the host cell and
proliferates. It is called "transformation" because the function of the
host cell may be altered. Normal E. coli cells are difficult to take up
plasmid DNA from the medium. If they are treated with CaCl2, the
transformation efficiency can be significantly enhanced. Even so, only
one cell in about 10,000 cells may take up a plasmid DNA molecule.
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c) Selective amplification. A specific antibiotic is added to kill E. coli

without any protection. The transformed E. coli is protected by the
antibiotic-resistance gene whose product can inactivate the specific
antibiotic. In this figure, the numbers of vectors in each E. coli cell are
not the same, because they may also reproduce independently.
d) Isolation of desired DNA clones.
DNA cloning using lamda phages as vectors:
Figure: Schematic drawing of the DNA cloning using l phages as vectors.
The DNA to be cloned is first inserted into the l DNA, replacing a

nonessential region. Then, by an in vitro assembly system (described
below), the l virion carrying the recombinant DNA can be formed. The l
genome is 49 kb in length which can carry up to 25 kb foreign DNA.
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Polymerase Chain Reaction (PCR) :Primers are in green color:
Materials required:
Two primers, each about 20 bases long with sequence
complementary to the sequence immediately adjacent to the DNA
segment of interest.
DNA polymerase (e.g., Tag polymerase) which can sustain high
temperature (> 60o C).

A large number of free deoxynucleotides (dNTPs).
The target DNA fragment.
Procedure:
Heat denaturation at about 95oC.
Primers bind to the denatured DNA by base pairing as the temperature
is gradually cooled to about 60o C.

Extend primers with Tag polymerase.
Repeat the above process. The number of copies doubles in each
cycle.
Typically 20 to 30 cycles are sufficient for effective DNA amplification.
Advantages:
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Much faster than using vectors.

Only very small amount of target DNA is needed.
Disadvantages:
To synthesize primers, we need to know the sequence flanking the DNA
segment of interest.
Applies only to short DNA fragments, typically less than 5 kb.
Preparation of a DNA Library:
DNA library is a collection of cloned DNA fragments. There are two types
of DNA library:
The genomic library contains DNA fragments representing the
entire genome of an organism.

The cDNA library contains only complementary DNA molecules
synthesized from mRNA molecules in a cell.

Genomic Library:
The genomic library is normally made by l phage vectors, instead of
plasmid vectors, for the following reasons:
The entire human genome is about 3 x 109 bp long while a plasmid
or l phage vector may carry up to 20 kb fragment. This would require 1.5
x 105 recombinant plasmids or l phages. When plating E. coli colonies on
a 3" petri-dish, the maximum number to allow isolation of individual
colonies is about 200 colonies per dish. Thus, at least 700 petri-dishes
are required to construct a human genomic library. By contrast, as many
as 5 x 104 l phage plagues can be screened on a typical petri-dish. This
requires only 30 petri-dishes to construct a human genomic library.
Another advantage of l phage vector is that its transformation efficiency is
about 1000 times higher than the plasmid vector.

cDNA Library:
The advantage of cDNA library is that it contains only the coding
region of a genome. To prepare a cDNA library, the first step is to isolate
the total mRNA from the cell type of interest. Because eukaryotic mRNAs
consist of a poly-A tail, they can easily be separated. Then the enzyme
reverse transcriptase is used to synthesize a DNA strand
complementary to each mRNA mlecule. After the single-stranded DNA
molecules are converted into double-stranded DNA molecules by DNA
polymerase, they are inserted into vectors and cloned.
Preparation of the genomic library:
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Figure: Preparation of the genomic library using l phage vectors. It is

basically the cloning of all DNA fragments representing the entire
genome.
Biotechnology firms
The top 10 publicly-traded biotechnology companies, ranked by 2003
sales, are:
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
X.
Amgen
Genentech
Serono
Biogen Idec
Chiron
Genzyme
MedImmune
Gilead Sciences
Cephalon
Millennium Pharmaceuticals
Schematic diagram of recombinant DNA technology is given

bellow:
How can you produce a protein of pharmaceutical interest?
Taking tissue
bacterial cell
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Taking
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Extraction of total RNA

plasmid DNA by
Extraction of
alkali
hydrolysis
Preparing cDNA by reverse transcription

restriction enzyme
Cutting with
using reverse transcriptase enzyme

Doing electrophoresis
to get appropriate
Taking a small volume of cDNA and amplify
plasmid
by PCR using specific primers of target DNA
Doing agarose gel electrophoresis to isolate

the target DNA
Cutting the DNA with restriction enzyme to

get appropriate size
Doing agarose gel electrophoresis to isolate

the target fraction
Target DNA after cutting with restriction

enzyme
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Mixing the target DNA with plasmid in presence of DNA lygase (Lygation)
Recombinant plasmid containing target gene
Transformed into host cells by E.coli
Culturing in an appropriate growth media
Production of target protein
Isolating and purifying the protein by different techniques such as

Extraction, precipitation, chromatography etc
Measuring the amount of protein by different methods such as Lawry

method
Now formulation of proteins using appropriate excipients
Dispensing into unit dosage form.
Roles of Pharmacist in Pharmaceutical Biotechnology:

Pharmacists are health professionals who assist individuals in making
the best of medication.
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The rapid expansion in the field of biotechnology products has

presented new opportunities for the pharmacist while creating a new
set of responsibilities.
The roles and opportunities of pharmacist in Biotechnology are
given below:
1. Pharmacists expand their role in clinical pharmacy services, clinical
research, drug distribution and drug information.
2. Pharmacists keep abreast of innovation in these areas including the
development of the new drug delivery systems.
3. The development of artificial organs like liver, kidney offering a new
responsibility.
4. The development of the blood purification systems has offered a
new responsibility.
5. Pharmacist should promote sound scientific judgment in selecting
new agents for formulary inclusion.
6. Pharmacist work in gene therapy.
7. Pharmacists have offered a responsibility in the development of
genetically engineered vaccines.
8. Pharmacists have responsibility in basic biotechnology research and
applied industrial research.
9. Pharmacists should have knowledge about recombinant DNA,
hybridomas and other techniques.
List of Biotech product:

Seri
Generic
Product
Company
al
name
name
name
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Indication
Approval
date
Page -20
No
1
Human
Humulin
Eli Lily
Insulin
2
3
Someterm
Digoxin
Subcutaneous administration for
Oct-1982
the treatment of diabetes mellitus

Protropin
Digibind
immune-Fab
Genetech
Burroughs
type-I and type-II.

Multiple scelorosis
Indicated for treatment of
Oct-1985
April-
wellcome
potentially life threating digoxin
1986
Schering-
intoxication
Viral infections and cancers
June-1986
Vaccinations against infection
Julay-
Interferon--
Intron A
2b
Hepatitis-B-
Recombiva
vaccine
somatotropin
x HB
Humatrop
Eli Lily
caused by Hepatitis-B-virus
Children with growth failure
1986
March-
Alteplase
e
Activase
Genetech
Inadequate assessed in ischemic
1987
Nov-1987
Heamophilus
Hib Titer
Praxis
stroke.
Vaccination against invasive
Dec-
Biologics
disease
19888
B-conjugate
plough
Merk
9
10
vaccine
Epoietin-
Interferon--
Epogen
AlferonN
Amgen
Interferon
Chronic renal insufficiency

Melanoma and essential
June-1989
Oct-1989
11
n3
Interferon--
Actimmun
sciences
Genetech
thrombocytes
Basal cell carcinoma cancer
Dec-1990
12
13
14
Ib
Filgrastim
Epoitin-
Aldesleukin
e
Neupogen
Procrit
Proleukin
Amgen
Ortho biotech
Cetus
Neutropenia
Chronic renal failure
Metastatic malignant melanoma,
Feb-1991
Feb-1991
Jun-1992
15
Darbepoetin-
Aransep
Kirin
renal carcinoma
Chronic renal insufficiency
Sep-2001
16
17
18
Bosentan
Anakinra
Frovatriptan
Tracleer
Kineral
Frova
Genetech
Amgen
Elan
Exercise ability hypertension

Active rheumatoid arthritis
Migraine attaks in adult
Oct-2001
Nov-2001
Nov-2001
Succinate
pharmaceutic
als
INDUSTRIAL MICROBIOLOGY
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Industrial microbiology or microbial biotechnology encompasses the

use of microorganisms in the manufacture of food or industrial products.
The use of microorganisms for the production of food, either human or
animal, is often considered a branch of food microbiology. The
microorganisms used in industrial processes may be natural isolates,
laboratory selected mutants or genetically engineered organisms.

Industrial microbiology is a field of microbiological sciences concerned
with the utilization of microorganisms in industrial processes. It deals with
all forms of microbiology having economic aspects. It is concerned with
biochemical conversion with the help of microbes.

Difference between biochemical and chemical processes:
Biochemical processes are based on the ability of microbes to
synthesize their own catalysts (i.e. enzymes) and the mass of microbes
increase with the progression of biochemical reactions.

The biochemical processes involve relatively low concentrations of
both substrate and products.

Biochemical reactions are restricted to aqueous phase and the
conditions of temperature and pH are usually mild

Maintaining the stability of biochemical conversion is more difficult
than chemical processes.

Chemical processes mainly concern with the purity of reactants,
whereas biochemical processes are featured with free from microbial
contamination, aseptic processing, sterilization and maintenance of
sterility.
What is fermentation?
Fermentation may be defined as the process of growing a culture of
microorganisms in a nutrient media and thereby converting feed into a
desired end product. It is also described as a biochemical reaction in
which microorganism (bacteria or fungi) serve as biocatalyst.

An aerobic (without oxygen) cellular process in which organic foods are
converted into simpler compounds and chemical energy (ATP) is
produced.
Fermentation occurs in fruits, bacteria, yeasts, fungi, as well as in
mammalian muscles.
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Fermentation based commercial products:

1) Food and beverages:
FOODS
Examples
Micro-organisms
Cereals, rice
Bacillus cereus
Egg and egg products
Salmonella
Raw fruits and vegetables Parasitic protozoa, shigella
Fish
Vibrio parahaemolyticus
Chess
Staphylococcus aureus
Dry milk
salmonella
BEVERAGES
Examples
Micro-organisms
Beer, wine, rum, brandy,
Saccharomyces
whiskey
cerevisiae
2) Antibiotics:
Examples
Tetracycline
Streptomycin
penicillin
Micro-organisms
a. Staphylococcus
b.
a.
b.
a.
b.
aureofaciens
Staphylococcus rimosus
Streptomyces griseus
Streptomyces galbus
Penicillium notatum
Penicillium
chrysogenum
Streptomyces
Clavulanic acid
clavuligerus
3) Vitamins:
Examples
Riboflavin
Ascorbic acid
Vitamin B12
a.
b.
a.
b.
Micro-organisms
Clostridium butyricum
Clostridium roseus
Acetobacter xylinum
Acetobacter
suboxydans
Pseudomonas
dentifrices
4) Amino acids:
Examples
L-lysin
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Micro-organisms
Corynebacterium
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MSG
glutamicium
Corynebacterium
Glutamic acid
glutamicium
Corynebacterium
glutamicium
5) Organic acids:
Examples
Citric acid
Glucoronic acid
Acetic acid
Lactic acid
Micro-organisms
Aspergillus niger
Aspergillus niger
Acetobacter spp
Lactobacillus delbrueckii
6) Alcohol:
Examples
Ethanol
Ethyl alcohol
Micro-organisms
Saccharomyces cerevisiae
a. Torula cremoris
b. Torula lactose
7) Carbohydrates:
Examples
Dextran
Micro-organisms
Leuconostoc
Xanthan gum
mesenteroides
Xanthomones compestris
Examples
Amylase
Cellulose
Invertase
Lipase
Protease
Micro-organisms
Aspergillus oryzae
Trichoderma reesii
Saccharomyces cerevisiae
Saccharomyces lipolytica
Bacillus
8) Enzymes:
9) Solvents:
Examples
Acetone butanol
2,3-butanedial
Dihydroxy acetone
10) Single cell proteins:
Examples
Methane
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Micro-organisms
Clostridium
acetobutylicum
Bacillus polymyxa
Gluconobacter suboxydans
Micro-organisms
Methylomonas
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Carbon-dioxide
11) Biological pesticides:
Examples
Bioinsecticides
methanoxidans
Spirulina maxima
Micro-organisms
a. Bacillus thuringiensis
b. Bacillus popilliae
What is fermenter?
Fermenter is a container in which a favorable environment is
maintained to operate a desired biological process. It is considered as
a heart of the fermentation process.

Simply, a fermenter is a vessel in which reagents, substrates and
organism are brought into contact with provision of their addition and
removal. Fermenter also called bioreactor.

Types of fermenter:
A) According to mode of operation:
1) Batch type:
Fermentation proceeds for defined period and terminated when the
product concentration reaches a preselected level.
2) Continuous type:
Fermentation is carried out by continuous feeding off sterile
medium and the continuous recovery of the medium containing
desired end products.
B) According to the culture methods:
1) Submerged fermenters (suspended-growth systems) organisms
are immersed in and dispersed throughout their nutrient medium.
2) Surface fermenters (supported-growth systems) organisms
grow as a layer or a film on a surface in contact with a nutrient
medium.
Factors or Steps involved in the design of a fermenter:

The following steps are involved in the design of a fermenter:
1. Selection of microorganism-it determines the phase of growth, pH,
the degree of aeration required etc.
2. Selection of fermenter configuration:
After selecting the micro-organism, the next step is the selection of
an appropriate fermentor configuration. Hence one should clearly
understand the merits and limitations of different fermenter such as
batch stirred-tank fermentor, continuous- stirred tank fermentor or
tubular fermentor etc.
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3. Determination of the fermentor dimensions:

The size of the fermentation batch governs the volume and
diameter of the fermentor and the type of fermentation determines
the values of the operating variables such as concentration,
temperature and PH.
4. Determining the extent of the heat transfer surface and
requirements of other devices such as power supply, aeration, and
agitator.
5. Selection of materials of construction
6. Mechanical design and the devices for the maintenance of aseptic
conditions.
7. Facilities for monitoring and control.
8. Safety factors.
Characteristics of an ideal fermentor:
1. The material of construction of a fermentor should be robust enough to
with withstand pressure.
2. It should be resistant to corrosion and free from any toxic effect for the
microbial culture.
3. The materials in the fabrication of, or lining of fermentation vessels
include copper, stainless steel, iron and glass.
Stainless steel or glass is not generally used for lining of fermentors,
because they are relatively more expensive. Sometimes wood also be
used.
4. The fermentation tank should be provided with a stirring device for the
uniform distribution of air, nutrients and microbes. The baffles should
be provided to avoid vortex formation.
5. In case of aerobic submerged fermentations, the tank should be
equipped with the aerating device.
6. It should be equipped with a sampling valve for withdrawing samples
for in-process laboratory tests.
7. It should contain a drain at the bottom for the removal of the
completed fermentation product.
8. It should have manhole at the top to gain access inside the fermentor
for different purposes, such as cleaning, repairing etc.
9. There should be a provision for controlling the temperatures and PH.
10.
A fermentor should permit easy control of contaminating microbes.
11.
There should be a facility for the intermittent addition of an
antifoam agent.
12. There should be provision for feeding certain medium components
during the progress of fermentation e.g. in penicillin fermentation.
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A typical fermentor with its auxiliary equipment:
Figure: typical fermentor with its auxiliary equipment
What is an antibiotic?
The word "antibiotics" comes from the Greek anti (against) and bios
(life). Antibiotics are drugs or chemical substance which derived from a
mold or bacterium that can kill microorganisms and cure bacterial
infections. e.g. Penicillium notatum.
Antibiotics that kill bacteria are called "bactericidal" and the ones
that stop the growth of bacteria are called "bacteriostatic".
Classification of antibiotics:
A simple classification of antibiotics is given below:
CLASS
-lactam
antibiotics
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ANTIBIOTICS
Penicillin, Amoxicillin, Ampicillin, Cloxacillin,
Cephalosporin, Nocardicins, Thienamycin,
Clavulanic acid.
Page -27
Aminoglycosides
Macrolides
Amikasin, Streptomycin, Gentamycin,

Kanamycin, Netilmicin, Tobramycin, Neomycins.
Tetracycline, Oxytetracycline, Minocycline,
Doxycycline, Aureomycin.
Erythromycin, Azithromycin, Clarithromycin,
Fluoroquinolones
Ansamycins
Polyenes
Cyclic
bacitracin.
Ciprofloxacin, Enoxacin, Norfloxacin, Ofloxacin.
Rifampicin, Maytansine.
Amphotericin B, Griseofulvin, Nystatin.
Polymixins, Gramicidin.
Tetracyclines
polypeptides
Miscellaneous
Chloramphenicol, Adriamycin, Mitomycins,

Clindamycin, Cycloserine.
Structure of Pen-V, Pen-G, Ampicillin and Amoxicillin:
How can we produce penicillin by fermentation technology?
The term penicillin is the generic name for a family of related

substances, which differ in the nature of side chain, attached to the basic
fused -lactam thiazolidine ring system. Penicillin production involves:
1) Method:
Penicillin may be produced by either
a. Surface culture method: in which the mold is grown on the
surface of shallow layers of the fermentation medium.
b. Submerged culture method: in which the mold is grown
submerged in the fermentation medium in shake flaks or deep
tank.
2) Mold:
Several strains are used in the production of penicillin either
Penicillium notatum or Penicillium chrysogenum. But carefully selected
stain of Penicillium chrysogenum is considered to be the best.
3) Master stock culture:
the selected production strain of Penicillium chrysogenum is
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maintained in the form of a master. This stock culture can be

preserved by
a. Freeze drying (lyophilization)
b. Fixing the spores in sterilized solid-sand mixtures.
c. Storage under liquid nitrogen i.e. in a frozen state.
4) Media:
The media used in the production of penicillin can be divided into:
a. Seed stage media:
Seed stage media are generally based on corn-steep liquor.
b. Production stage media:
Production stage media are based on
Cornsteep
One or more sugar sugars like lactose, sucrose and
glucose.
Mineral salts including sources of sulphate and
phosphate.
The requisite precursor,
e.g. phenyl acetic acid or phenoxy acetic acid.
5) Inoculation method:
The various media employed in the manufacture of penicillin can be
inoculated by several methods:
a. In surface culture methods, the surface medium is inoculated
with dry spores. The spore material is applied in such a way as to
cover the surface as uniformly as possible.
b. In submerged culture methods, the production medium is
inoculated with dry spores, by pellet inocula or un-germinated
spores in suspensions.
6) Raw materials:
For the production penicillin the raw material is selected on the basis of
a. Nutritive requirements of the mold.

b. The conditions for the optimum accumulation of penicillin.
c. Subsequent extraction and purification of the penicillin.
Lactose in a concentration of 6% is very satisfactory source of
carbon while sodium nitrate, ammonium sulphate, ammonium acetate,
ammonium lactate, Cornsteep liquor etc serve as source of nitrogen.
7) Condition of fermentation:
a. A temperature of about 250C appears to optimum.
b. Control of PH range 5 7.5 is important.
c. Calcium carbonate used as buffering agent.
d. Penicillium chrysogenum being strictly aerobic adequate
aeration of the temperature is essential.
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e. The effectiveness of the aeration can be enhanced by use of

pressure.
f. Antifoam agents such as tributyl citrate, octadecanol and lard
oil prevents excessive foam formation during the production of
penicillin.
g. Prevention of contamination during the penicillin production
is essential.
8) Duration:
The duration of penicillin production either
Short (120 140 hours)
Extended (180 240 hours)
9) Isolation and purification:
The first step in the recovery process is the removal of mycelium or
cells by filtration or centrifuging.
The second step is to remove the antibiotic from the spent
production medium by solvent extraction, adsorption or
precipitation.
Finally the antibiotic is tested as sayed and certified as specified in
the relevant pharmacopoeia.
Flow diagram for the production of penicillin-G:
A schematic flow diagram for the production of Penicillin-G is shown
below:
Master stock of Penicillium chrysogenum
Sporulation (flask culture)
Spore suspension
Germinator/seed tank
Production fermentor
Filtration
Filtrate
Solvent extraction
(Butyl acetate/Amyl acetate)
Rich solvent
Aqueous extraction
(Potassium bicarbonate/acetate)
Rich aqueous solution

Active carbon treatment
Ion-exchange
Adsorption/elution
as potassium salt
Azeotropic distillation
Filtration/solvent wash
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Crystalline mass
Drying blending
Potassium penicillin-G Bulk
(Non-sterile)
IMMUNOLOGY
What is immunology and immunity?
Immunology is the study of immune system, the development of
vaccines and the regulatory produce for manipulating the immune
response.
Immunity means the capacity of the body to resist infection or to resist
the invasion of foreign particles or micro-organisms that tend to
damage our tissues.

Types of Immunity:
Immunity
Natural immunity
Specie
Racial Individual
Acquired immunity
Active
Passive
Natural
Artificial
Natural
Artificial
Clinical or
Subclinicaldisease
Vaccines:
Congenital
a. Toxoids
Colostrum
b. Suspensions of
microorganisms
Antiserum
Antitoxin
-globulin
A) Natural immunity:
This is resistance to disease possessed as part of an individuals
constitutional make-up. It results in differences between species, races
and individuals.
1. Species:
Some diseases like tuberculosis, anthrax, psittacosis and rabies etc
occur both in animals and man alike. Man is susceptible to plague but
fowls are not. Mice are not affected by typhoid fever but it is a serious
disease in humans.
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2. Race:
Negroes have a high resistance to yellow fever but white men are
very susceptible.
The Caucasian race is more resistant to tuberculosis than Negroes
or American Indians.
3. Individuals:
Some people are more resistant than others to colds and skin
infections. Most of the children (2 to 5 years) are susceptible to
diphtheria whereas most adults are immune to it.
B) Acquired immunity:
Natural immunity is inadequate for protection against many
microbial diseases and during lifetime additional immunity is acquired
either actively, due to stimulation of the individuals antibody-producing
cells (active immunity) or passively, as a result of the introduction of
antibodies from another person or animal (passive immunity).
1) Active immunity:
It may be required either naturally or artificially.
1. Naturally acquired active immunity:
a. Clinical infection:
When a patient recovers from certain diseases he is left with a
high degree of immunity. In the case of diphtheria, smallpox and
poliomyelitis, for example, this persists for life. For a number of
disease, however, the immunity is of short duration e.g. after
influenza, pneumonia and gonorrhoea.
b. Sub-clinical infection:
This type of immunity may also be acquired after subclinical
infection due to smaller number of invading organisms. A person
becomes immune because his antibody producing cells have
received an adequate stimulus. Thus children and adults staying in
slums develop naturally acquired active immunity to a variety of
diseases as they are frequently exposed to sub-infection.
2. Artificially acquired active immunity:
This type immunity is acquired by the administration of antigens
usually by injection. The antigens used in this way are known as
vaccines and may be alive or dead microorganisms or their products.
Vaccines are of two types
a. Toxoids:
These are bacterial exotoxins modified, so that their toxicity is
destroyed or reduced to a safe level.
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b. Suspensions of microorganisms:
These consist of microorganisms that have either been killed
or if this treatment affects their antigenicity (i.e. capacity to
stimulate antibody- production), attenuated.
2) Passive immunity:
It may also be required either naturally or artificially.
1. Naturally acquired passive immunity:
Antibiotics for diseases to which the mother is immune may be
transmitted to the foetus via the placental blood and give immunity to
the infant for several months. For example, babies show high
resistance to chickenpox, diphtheria, measles and scarlet.
2. Artificially acquired passive immunity:
This type of immunity is acquired by injecting the preparations
known as antisera, sera or immune sera and the official products
include preparations containing
a. Antitoxic antibodies: antibodies that neutralize exotoxins.
Botulinum antitoxin
Diphtheria antitoxin
Gas-gangrene antitoxin (oedematiens)
Gas-gangrene antitoxin (septicum)
Gas-gangrene antitoxin (welchi)
Tetanus antitoxin
b. Antibacterial antibodies: antibodies that combat endotoxin
producing bacteria
Lepto-spira antiserum
c. Antiviral antibodies: antibodies that combat viruses.
Rabies antiserum
What is infection?
When microorganisms successfully invade the body and cause
damage to the tissues, infection is said to be occurred. Consequently,
diseases produced by micro-organisms are called infectious diseases.
Ways of spreading infection:
1. Physical contact with a diseased person or animal
2. Droplet infection
3. Dust-borne infection
4. Contact with contaminated articles
5. Hand infection
6. Arthropod infection
1) Physical contact with a diseased person or animal:
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As the causal organism has only a brief existence outside the human
body, venereal diseases are almost always transferred in this way.

Rabies is transferred to man through the bite of an infected dog.
2) Droplet infection:
Coughing, sneezing and quiet talking cause expulsion of fine droplets
from the respiratory tract containing microorganisms which may be
inhaled by individuals nearby.
Respiratory diseases are often transmitted by this mechanism.
3) Dust-borne infection:
Some disease producing bacteria, e.g. Mycobacterium tuberculosis,
remain alive for long period of time in the dried condition, and
contaminated dust stirred up by cleaning process may infect others.
4) Contact with contaminated articles:
Clothes, bedding, handkerchiefs, towels, toys and other items
recently contaminated by a diseased person are source of infection.
5) Hand infection:
The hands are potential means by which microorganisms from
saliva, nasal mucus, skin infections, and even feces are transferred to
other individuals and to food.
6) Arthropod vectors:
Some disease-producing organisms are transmitted by insects (e.g.
Flies and mosquitoes) and other arthropods (e.g. Fleas and ticks).
In certain cases the vectors simply acts as accidental carrier of the
organisms from filth to food or to the human host. In this way, plague
and typhoid fever are transferred by fleas and house flies respectively.
In other cases, the microorganisms are transmitted by blood-sucking
arthropods and spend their part of life cycle in the vector.

Examples are the transmission of malaria by the Anopheles mosquito,
yellow fever by the Aedes mosquito, sleeping sickness by the tsetse
fly, and murine typhus by the rat flea.
Antigen
An antigen is a substance/molecule when introduced into the body
produces an antibody by the immune systems, which will then kills or
neutralize the antigen that is recognized as a foreign and potentially
harmful invader.
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Any substance (as a toxin or enzyme) that stimulates an immune

response in the body (especially the production of antibodies).
e.g. virtually all proteins, many polysaccharides, lipoproteins,
synthetic polypeptides and an enormous number of small molecules
that can be suitably linked to proteins: can act as antigen.

All antigens have two properties:
a. Immunogenicity: the capacity to stimulate the formation of the
corresponding antibodies.
b. Specificity: ability to react specifically with those antibodies.
Antibody:
An antibody, also known as an immunoglobulin, is a large Y-shaped
protein used by the immune system to identify and neutralize foreign
objects like bacteria and viruses.

A protein produced by the body's immune system that recognizes and
helps fight infections and other foreign substances in the body.
Chemically antibodies are immunoglobulin (protein molecules).

Hapten:
A hapten is a small molecule that can elicit an immune response only
when attached to a large carrier such as a protein; the carrier may be
one that also does not elicit an immune response by itself.

An incomplete antigen, typically a small molecular weight substance,
being incapable of causing the production of antibodies, but capable of
combining with a specific antibody.

Epitope:
An epitope, also known as antigenic determinant, is the part of an
antigen that is recognized by the immune system, specifically by
antibodies, B cells, or T cells.
The epitopes of protein antigens are divided into two categories,
conformational epitopes and linear epitopes

A specific site on an antigen that stimulates specific immune
responses, such as the production of antibodies or activation of
immune cells.
How can we produce antibody from plasma?
Immunize the mouse with specific antigen
Check the antibody level (titre)
When the antibody level in the blood is high dissect the mouse / animal
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Collect spleen
collect the blood from the
heart
Isolates spleen cells (-cells)
Fudge with myeloma cell in presence of PEG
Centrifugation
Collect plasma
(antisera), it contains
Cell fusion
Produce hybridoma cells
produce polyclonal
antibodies
Grow in HAT (Hypoxanthene aminopterinethymidine) medium
Cloning of hybridoma cell to get single cell (-cells)
Culture
Produce monoclonal antibodies in the medium
ELISA (Enzyme linked immunosorbent assay) is done,
to check the monoclonal antibody productions
Collect the hybridoma cell from the dish
Inject into the abdominal cavity of female mouse
After few days collect the ascites fluid
Then purify ascites fluid with ammonium sulphate
(NH4SO4)
Centrifugation
Collect ppt and remove water
Dissolve ppt in buffer (which buffers, separates antibody from antigen).
Run through an affinity column chromatography
Wash this antibody
Measured the amount of protein by Bronsted-Lowry method
Get pure Antibody
Use the require dosage form, then ready for dispense.
Immunoglobulin:
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One of a group of proteins (globulins) in the body that act as

antibodies. They are produced by specialized white blood cells called B
cells and are present in blood serum and other body fluids. An antibody is
typically a Y-shaped structure consisting of four polypeptide chains two
heavy chains and two light chains.
Immunoglobulins are glycoprotein molecules that are produced by
plasma cells and that function as antibodies in the immune response by
binding with specific antigens. There are five classes of immunoglobulin:
IgA, IgD, IgE, IgG, and IgM.
Structure of immunoglobulin:
Figure: the structure of Immunoglobulin
Types of Immunoglobulin:
The five subclasses of antibodies are:
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1. Immunoglobulin-G (IgG):
The most abundant type of antibody is found in all body fluids. They
are smallest but most common antibody (75 80%) of all antibodies in
the body mainly in serum and lymph. There are four IgG subclasses
namely IgG-1, IgG-2, IgG-3 and IgG-4.
Function:
Protection against bacterial and viral infections, attaches to
phagocytes and tissues, protect her baby (fetus).
2. Immunoglobulin-M (IgM):
IgM antibodies are the largest antibody. They are found in blood and
lymph fluid and are the first type of antibody made in response to an
infection. which is found mainly in the blood and lymph fluid. IgM
antibodies are about 5% to 10% of all the antibodies in the body.
Function:
Protection against early infection, bacterial to gram negative
bacteria, complement fixation.
3. Immunoglobulin-A (IgA):
This is found as secretory antibodies in the tears, saliva,
gastrointestinal tract, colostrums and other secretions. About 10
15% of the antibodies present in the body are IgA antibodies. They are
also known as secretory antibodies (sIgA).
Function:
Protection to mucous membranes and internal cavities against
infection.
IgA antibodies protect body surfaces that are
exposed to outside foreign substances.

4. Immunoglobulin-D (IgD):
IgD antibodies are found in small amounts in the tissues that line
the belly or chest. It also found in serum and lymphocytes.
Function:
Controls antigen stimulation of -cell fetal antigen receptor.
5. Immunoglobulin-E (IgE):
IgE antibodies are found in the lungs, skin, and mucous membranes.
They cause the body to react against foreign substances such as

pollen, fungus spores, and animal dander. They may occur in allergic
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reactions to milk, some medicines, and some poisons. IgE antibody

levels are often high in people with allergies.
Function:
Combats parasitic diseases, causes allergies, drug sensitivity,
anaphylaxis and immediate hypersensitivity.
Monoclonal antibody-based therapeutics on the market :

Product name
Target antigen
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Therapeutic
Company
Page -39
(Substance
name)
Mabthere /rituxa
n
(Rituximab)
Herceptin
(Trastuzumab)
use
CD20
surfaceantigen
of B
lymphocytes
Human
epidermal
growth factorlike receptor 2
(HER2)
Remicade
(Infliximab)
TNF-alfa
Treatment of
Non-Hodgekins
lymphoma
Genentech/
Hoffmann LaRoche
Treatment of
metastatic
breast cancer
overexpressing
HER 2 protien
Genentech/
Hoffmann LaRoche
Treatment of
rheumatoid
arthritis
Centocor
ReoPro
(Abciximab)
Platelet surface
receptor GP
IIb/IIIa
Prevention of
blood clot
Centocor
Orthoclone
OKT3
(Muromomab)
T-lymphocyte
surface antigen
CD 3
Reversal of
acute kidney
transplant
rejection
Ortho Biotech
What is ELISA?
Enzyme-linked immunosorbent assay (ELISA), also known as an
enzyme immunoassay (EIA), is a biochemical technique used mainly in
immunology to detect the presence of an antibody or an antigen in a
sample. The ELISA has been used as a diagnostic tool in medicine and
plant pathology, as well as a quality-control check in various industries.

Types of ELISA:
There are four types:
1. "Indirect" ELISA
2. Sandwich ELISA
3. Competitive ELISA
4. Multiple and Portable ELISA (M&P ELISA)(ELISA Reverse in
published papers)
1) "Indirect" ELISA
The steps of indirect ELISA follow the mechanism below:-
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A buffered solution of the antigen to be tested for is added to each well

of a microtiter plate; where it is given time to adhere to the plastic
through charge interactions.
A solution of non-reacting protein, such as bovine serum albumin or

casein, is added to block any plastic surface in the well that remains
uncoated by the antigen.
Next the primary antibody is added, which binds specifically to the test
antigen that is coating the well. This primary antibody could also be in
the serum of a donor to be tested for reactivity towards the antigen.
Afterwards, a secondary antibody is added, which will bind the primary

antibody. This secondary antibody often has an enzyme attached to it,
which has a negligible effect on the binding properties of the antibody.
A substrate for this enzyme is then added. Often, this substrate

changes color upon reaction with the enzyme. The color change shows
that secondary antibody has bound to primary antibody, which strongly
implies that the donor has had an immune reaction to the test antigen.
This can be helpful in a clinical setting, and in R&D.
The higher the concentration of the primary antibody that was

presents in the serum, the stronger the color change. Often a
spectrometer is used to give quantitative values for color strength.
2) Sandwich ELISA:
A less-common variant of this technique, called "sandwich" ELISA, is used
to detect sample antigen. The steps are as follows:
1. Prepare a surface to which a known quantity of capture antibody is
bound.
2. Block any nonspecific binding sites on the surface.
3. Apply the antigen-containing sample to the plate.
4. Wash the plate, so that unbound antigen is removed.
5. Apply enzyme linked primary antibodies as detection antibodies
that also bind specifically to the antigen.
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6. Wash the plate, so that the unbound antibody-enzyme conjugates

are removed.
7. Apply a chemical that is converted by the enzyme into a color or
fluorescent or electrochemical signal.
8. Measure the absorbency or fluorescence or electrochemical signal
(e.g., current) of the plate wells to determine the presence and
quantity of antigen.
A sandwich ELISA.
a. Plate is coated with a capture antibody;
b. Sample is added, and any antigen present binds to capture
antibody;
c. Detecting antibody is added, and binds to antigen;
d. Enzyme-linked secondary antibody is added, and binds to
detecting antibody;
e. Substrate is added, and is converted by enzyme to detectable
form.
3) Competitive ELISA
A third use of ELISA is through competitive binding. The steps for this
ELISA are somewhat different than the first two examples:
1. Unlabeled antibody is incubated in the presence of its antigen
(Sample).
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2. These bound antibody/antigen complexes are then added to an

antigen-coated well.
3. The plate is washed, so that unbound antibody is removed. (The more
antigens in the sample, the fewer antibodies will be able to bind to the
antigen in the well, hence "competition.")
4. The secondary antibody, specific to the primary antibody is added.
This second antibody is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or
fluorescent signal.
4) Multiple and Portable ELISA (M&P ELISA)(ELISA Reverse in
published papers):
The entire device is immersed in a test tube containing the
collected sample and the following steps (washing, incubation in
conjugate and incubation in chromogenous) are carried out by dipping the
ogives in microwells of standard microplates pre-filled with reagents.
The advantages of this technique are as follows:
1. The ogives can each be sensitized to a different reagent, allowing
the simultaneous detection of different antibodies and/or different
antigens for multi-target assays
2. The sample volume can be increased to improve the test sensitivity
in clinical (saliva, urine), food (bulk milk, pooled eggs) and
environmental (water) samples
3. One ogive is left un-sensitized to measure the non-specific reactions
of the sample
4. The use of laboratory supplies for dispensing sample aliquots,
washing solution and reagents in microwells is not required,
facilitating the development of ready-to-use lab-kits and on-site kits.
What Is Serology? Types of serological reaction:

The study of antigen-antibody (Ag-Ab) reactions is called serology.
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The science that deals with the properties and reactions of serums,
especially blood serum.

The most common in-vitro serological tests include:
1. Agglutination
2. Precipitin
3. Complement fixation
4. Lysis by complement
5. Radioimmunoassay (RIA)
6. Enzyme-linked Immunosorbent Assays (ELISA)
7. Opsonization
1) Agglutination:
When suspensions of bacteria or red blood cells (Ags) are mixed with
their antisera (Abs) they are usually clumped (agglutinated). Agglutination
reaction is carried out in physiological salt solution. The ionic strength is
important because unless the net negative charge of bacteria is damped by
counter-ions, the cell cannot approach other closely enough for Ab molecules
to form specific bridges between them.
The agglutination technique is used in the:
VDRL (venereal disease research laboratory) and RPR (rapid
plasma re-agin) tests for syphilis.
Widal test for typhoid fever
Weil-Felix test for rickettsia disease and a test for pregnancy.
2) Precipitin:
The precipitin test is useful in detecting antibodies to the endotoxins of
tetanus, diphtheria and scarlet fever. It is also used to identify various serum
proteins in blood.
3) Complement fixation:
Complement fixation reaction is used to identify certain viral infections
such as small-pox, influenza and poliomyelitis.
Complement is fixed (bound) when antigens bind with antibodies, even
if the antigens are not on a RBC or bacterial cell.
4) Lysis by complement:
When the IgG and IgM antibodies are bound to antigens, the antibodies
become activated and react with complement; as result the complement is
activated. If the antigens are on cells such as RBCs or on pathogens such as
Vibrio cholera, the activated complement will lyse the cells.
5) Radioimmunoassay (RIA):
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Radioimmunoassay (RIA) is a sensitive, versatile technique, using

radioactively labeled antigen or antibody. It is frequently employed to
determine small amounts of drugs, hormones or antigens, such as hepatitis
B antigen in blood donor serum.
6) Enzyme-linked Immunosorbent Assays (ELISA):
Enzyme-linked Immunosorbent Assays (ELISA) is sensitive techniques
that use an enzyme-Ab-Ag combination absorbed onto the sides of a test
well. If the patient has Abs or Ags for the disease agent, the linkage is
formed.
Addition of the substrate for the enzyme causes a color change,
indicating a positive test result. If the patient does not have the serum
antigen or the antibody sought, the enzyme is not linked and no color
change is observed. These techniques are routinely used to test for AIDS and
other diseases.
7) Opsonization:
Opsonize means to engulf and the Opsonization test is based on the
fact that phagocytes can engulf more bacteria if the specific antibodies for
those bacteria are present. The test is seldom used in a clinical laboratory
but is valuable as a confirming test for bacterial diseases.
ATISENSE TECHNOLOGY
What is antisense technology?
Antisense technology is a nucleic acid based approach to down
regulate the expression of gene with the aim of preventing the
inappropriate or over expression derived disease state.

Various disease states are associated with the inappropriate or
overproduction of gene, for example :
a. The expression of oncogene, leading to cancer.
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b. The over expression of cytokines during some disease state, with

worsening of disease symptoms.
c. The over production of angiotensinogen which ultimately results in
hypertension.
d. If COX-2 is over expressed in prostate gland there is a chance of
prostate cancer.
Major classes of antisense agents:
There are three major classes of antisense agents1. Antisense oligonucleotides
2. Anti-gene sequence
3. Ribozymes
How does antisense technology works?
In antisense approach a short single stranded specific nucleotide
sequence termed as Antisense oligonucleotide is used. Antisense
oligonucleotides can be either DNA or RNA.

These oligonucleotides are capable of binding to DNA or more
commonly to mRNA derived from gene, this binding in most cases
occurs via Watson and Crick based pair complementary.
DNA (specific gene)
Transcription
AntisenseOligonucleotides
mRNA
Translation
Protein
Biological Effect
Binding prevents expression of gene product by preventing either the

transcription or the translation process.
C
Transcription
PROTEIN
Translation
A
A
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T
C
G
G
C
DNA
mRNA
Antisense oligonucleotide
Figure: Outline of how an Antisense oligonucleotide can present synthesis of

gene product by blocking translation .In practice, antisense oligos are 1218 nucleotides in length.
The prevention of mRNA translation by duplex formation (hybridization)

with Antisense oligonucleotides assumed to be carried out by twin
mechanism.
1. First the oligonucleotides acts as a steric blockers, prevents proteins
involves in translation.
2. The generation of duplex also allows the intracellular RNases such
as RNase H to target the duplex. This enzyme is capable of binding
to RNA-DNA duplex (most of the Antisense oligonucleotides are DNA
based).
Use of Antisense technology:
Antisense oligonucleotides (oligos) are assessed pre-claimed and
clinical studies in the treatment cancer, as well as a variety of viral
diseases such as HIV, hepatitis B, Herpes- virus and papillomavirus
infection.
Antisense technology has also potential application on restenosis,
rheumatoid arthritis and allergic disorder in which blocking of gene
expression may have a beneficial effect.
Advantages of Antisense technology:

0709BPM00188
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Specificity: Antisense oligos display extreme specificity binding with
the target mRNA.

Minimal toxicity: Most trials show that most Antisense technology
has few or no side effects. This is due to the highly specific nature of
oligo duplexing, and the fact that they are nature biomolecules.
Low dose: AS target mRNA is usually present only in nano-molar
concentration (nM), the oligonucleotide required only in low level.

Easy to synthesis: The ability to manufacture oligos of specified
nucleotide sequence is relatively straight forward using automated
synthesizers.
Disadvantage of Antisense technology:
Sensitivity to nucleuses.
Very low serum half-lives.
Poor rate of cellular uptake.
Orally inactive.
VITRAVENETM Injection
Drug description (Fomivirsen sodium intravitreal) :
Vitravene (fomivirsen sodium intravitreal injectable) is a sterile,
aqueous, preservative free, bicarbonate-buffered solution for intravitreal
injection.
Fomivirsen sodium is a phosphorothioate oligonucleotide, twenty-one
nucleotides in length, with the following sequence:
5' GCG TTT GCT CTT CTT CTT GCG 3'
Active: Fomivirsen sodium 6.6mg
Indication
1. Local treatment of cytomegalovirus (CMV) retinitis patients
with acquired immunodeficiency syndrome (AIDS).
2. Ocular infection caused by:
Syphilis
Candidiasis
Toxoplasmosis
Histoplasmosis
Herpes simplex virus
Varicella-zoster virus
Side effects:
Adverse experiences reported in approximately 5 to 20% of patients
have included Ocular:
Abnormal vision, Anterior chamber inflammation, Blurred vision,
Cataract, conjunctive hemorrhage, Decreased visual acuity,
0709BPM00188
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Desaturation of color vision, Eye- pain, Floaters, Photophobia,

Retinal detachment, Retinal pigment changes, uveitis.
Systemic:
Abdominal pain, Anemia, Asthenia, diarrhea, fever, headache,
infection, sepsis, systemic CMV, vomiting, asthenia.
Adverse experiences reported in approximately 2 to 5% of patients
have include
Ocular:
Application site reaction , conjunctival hyperemia,
conjunctivitis, corneal edema, decreased peripheral vision, eye
irritation, hypotomy, keratic precipitates, optic neuritis, photopsia,
retinal vascular diseases, visual field defect, vitreous hemorrhage,
vitreous opacity.
Systemic :
Abnormal liver function, abnormal thinking, allergic reaction,
anorexia, back pain, bronchitis, depression, kidney failure etc.
ENZYME IMMOBILIZATION
What is enzyme immobilization?

Immobilization defined as imprisonment of an enzyme in a distinct
phase that allows exchange with, but is separated from the bulk phase in
which the substrate, effector or inhibitor molecules are dispersed and
monitored.
Advantages of immobilized enzymes:
There are many potential advantages of using immobilized enzymes.
They are:
1. Reuse:
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The same enzyme can be used repeatedly, since they are not lost at the
end of a batch.
2. Continuous use:
Continuous production system can be designed easily.
3. Cost effectiveness:
If the enzymes are immobilized, they can be used continuously or
repeatedly and hence cost effectiveness is achieved.
4. Less contamination:
Since the immobilized enzyme remains within the polymer matrix, it will
not contaminate the final product.
5. Stability:
Immobilization increases the thermal stability of the enzyme. For
example, immobilized glucose isomerase is stable at 650C for almost one
year, whereas the pure enzyme is denatured within few hours at a
temperature of 450C.
6. Process:
Since the immobilized enzyme have standardized activity, the process
control becomes very easy.
7. Enzyme substrate ratio:
It is very high in immobilized enzymes and this increases the cost
effectiveness.
Methods of enzyme immobilization:

There are two different methods
1. Immobilization in a support
2. Immobilization on a support
1) Immobilization in a support:
a) Entrapment:
In this technique, the enzyme is entrapped within a cross-linked
polymer matrix. Enzyme is dissolved in a solution of precursors of the

polymer and then polymerization is initiated. The enzyme is physically
entrapped within the matrix and it cannot escape by permeation. The
common polymers used include polyacrylamide gel, starch gel, silicon
rubber gel, cellulose triacetate, alginate, gelatin and agar.
0709BPM00188
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Figure: Entrapment
b) Microencapsulation:
In this method the enzyme immobilized by enclosing a droplet of
enzyme in a semi-permeable membrane capsule. This method has wide
applications in pharmaceutical and medicinal fields. Once encapsulation
is done, the enzyme cannot escape, whereas the low molecular weight
substrates and products can diffuse through the membrane.
The capsule may be made up of either permanent material like polylactic acid or phospholipid liposomes.
Figure: Encapsulation
2) Immobilization on a support:
a) Adsorption:
Adsorption of the matrix on the polymeric matrix is the easiest
method of immobilization. The adsorption occurs due to nonspecific
bonding like electrostatic, hydrophobic or affinity bonding to specific
ligand.
The disadvantage of this method is the loss of activity of enzyme
during adsorption. To minimize it, adsorbents should be chosen e.g.
alumina, amberlite CG-50, bentonite, calcium phosphate gel, carboxy
0709BPM00188
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methyl cellulose (CMC), collagen, glass, silica gel etc.
Figure: Adsorption
b) Covalent bonding:
The enzyme can be covalently bound to the polymer by many methods.
The forms a covalent bond with the active groups on a polymer support.
This can be done by two ways:
i. Through the reactive group on the side chains of its amino acids such
as lysine, arginine and tyrosine.
ii. Through the terminal amino and carboxyl groups of polypeptide
chains.
Figure: Covalent bonding
c) Cross linking:
Cross linking of the enzyme to itself by the use of a bi-functional
reagent with the inclusion of an inert protein in another important
0709BPM00188
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technique of immobilization. The most commonly used bi-functional

reagent is gluteraldehyde. The method is cheap and simple.
Figure: Cross linking
Three techniques are used immobilization of enzymes by cross linking.

They are: 1. Cross-linking of the enzyme with gluteraldehyde to form an
insoluble aggregate.
2. Adsorption of the enzyme onto a surface followed by cross-linking.
For example, the enzyme can be cross linked and adsorbed onto the
surface of particles like colloidal silica.
3. Impregnation of porous matrix material with the enzyme and crosslinking the enzyme within the pores. The common porous matrix
used in collodion membrane.
Applications of enzyme immobilization:

The enzyme applications are broadly classified into four categories
namely1. Medical uses.
2. Analytical uses.
3. Manipulative uses.
4. Industrial uses.
0709BPM00188
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1) Medical uses:
The rapidly growing area of medicine now involves the use of free or
extracellular enzymes. They are:
Enzymes are used to treat certain inborn errors of metabolism

which occur due to a missing enzyme.
Penicillin allergy in humans can be treated by injecting penicillinase

solution.
Enzymes are important in detection of infectious diseases.

Amylase, lipase and protease enzymes are used to correction of
digestive disorders.
Thrombins are used to stop bleeding during surgeries and tooth

extraction.
Glucose oxidase is used for diagnosis of sugar level in blood and

urine.
Lysozyme is used in the treatment of certain ulcers, measles,

multiple sclerosis, and some skin diseases.
Hyaluronidase is used to enhance the diffusion of antibiotics,

adrenaline, heparin, and local anesthetics in dentistry and surgery.
Streptokinase is used as anti-inflammatory agent.

2) Analytical uses:
Enzymes in free form as well as in immobilized form are making
important contributions in analytical methods involved in biochemistry
and medicine. They are:
Immobilized enzyme electrode permits continuous monitoring of

small concentrations of a specific biochemical.
Using immobilized enzyme electrodes, biochemical tests can be

automated.
Immobilized enzyme electrodes have been constructed for a variety

of biologically important compounds such as acetaldehyde, Dalanine, L-arginin, L-cysteine.
Important application of enzymes is in biosensors which displaying

characteristic specificity with chemical or electronic sensor to
convert a biological compound into electronic signal.
0709BPM00188
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Invertase enzyme is used to conversion of glucose into fructose.

Penicillin acylase is used in the production of semisynthetic
penicillins.
Ribonuclease is used to nucleotide production from RNA.

Asparatase is used in production of L-asparatic acid.
3) Manipulative uses:
Many enzymes isolated from different sources are applied in genetic
engineering as biological tools. These are:
Restriction endonucleases are used for isolating required genes,
mapping long strands of DNA and analyzing the chromosome
structure and other purposes.
S1 nuclease is used in conversion of cohesive ends into blunt ends.
DNA ligase seals the signal strand nicks in DNA which have 5-> 3OH termini. There are two extensively used DNA ligases- one from
E.coli and the other from T4 bacteriophage.
Restricted plasmid is treated with alkaline phosphatase enzyme.
The enzyme digests 5 phosphoryl group.
Reverse transcriptase enzyme is used in the synthesis of cDNA from
m-RNA. This enzyme is highly useful in construction of cDNA cline
bank.
DNA polymerases are used in synthesis of cDNA template. They also
catalyze
a 5->3 and 3->5 exonucleolytic degradation of
DNA.
4) Industrial uses:
Enzymes are used in different industries for different
purposes. These are:
Amylase enzyme id used in the textile industry to remove starch.

Proteolytic enzymes are used in softening of skin in leather industry.
Endoxylanase is used for bleaching pulp in paper industry.
Renin, catalase and lipase are used in the manufacturing of cheese.
0709BPM00188
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Pectinase is used to make fruit juice and wine clear.

Invertase is used in the manufacturing of chocolate covered
candies.
Glucose isomerase is used in soft drink industry.

Lactase is used in ice-cream industry.
GENE THERAPY
What is gene therapy?
Genes; those are carried on chromosomes, are the basic physical and
functional units of heredity.
Gene therapy is a technique for correcting defective genes responsible

for disease development.
Gene therapy can be described as the intracellular delivery of genetic

material to generate a therapeutic effect by correcting an existing
abnormality or providing cells with a new function.
Types of gene therapy:

There are two types of gene therapy. They are1. Germ-line or heritable gene therapy.
2. Somatic-cell or nonheritable gene therapy.
1) Germ-line or heritable gene therapy:
The technology of germ-line gene therapy is relatively simple and
requires no targeting as genetic abnormalities can be corrected by direct
manipulation. With increasingly sophisticated techniques being developed
in the field of transgenic animals, it is now possible, using homologous
recombination, to replace old genes for new.
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The results of the human genome project and establishment of

gene function will open up new possibilities for genetic intervention that
could be passed down through generations.
2) Somatic-cell or nonheritable gene therapy:
Somatic gene therapy involves the insertion of genes into diploid
cells of an individual where the genetic material is not passed onto its
progeny. The transfer is currently achieved by use of lipids and virusmediated systems. To date, there are three divisions of somatic gene
therapy. They areI. Ex vivo delivery.
II. In situ delivery.
III.In vivo delivery.
Vectors used in gene therapy:

In general, a gene cannot be directly inserted into a persons cell. It
must be delivered to the cell using a carrier or vector. Vector systems can
be divided into:
1. Viral vectors.
2. Non-viral vectors.
A) Viral vectors:
Many viral vector systems now exist for use in gene therapy. The most
widely used include1.
Retroviral vectors.
2.
Adenoviral vectors.
3.
Adeno-associated viral vectors.
1) Retroviral vectors:
Advantages:
Integration into host DNA.

All viral genes removed.
Relatively safe.
Disadvantages:
Semi-random integration.
0709BPM00188
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Transduction requires cell division.

Relatively low titer.
2) Adenoviral vectors:
Advantages:
Higher titer.
Efficient transduction of nondividing cells in vitro and in
vivo.
Disadvantages:
Toxicity.
Immunological response
Prior exposure.
3) Adeno-associated viral vectors:

Advantages:
All viral genes removed.

Safe.
Transduction of nondividing cells.
Stable expression.
Disadvantages:
Small genome limits size of foreign DNA.
Labor-intensive production.
Status of genome not fully elucidated.
B) Nonviral vectors:
An alternative to the viral strategies has been the application of
chemically synthesized vehicles such as liposomes.
1) Liposomes:
Advantages:
Absence of viral components.

Lack of previous immune recognition.
Disadvantages:
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Inefficient gene transfer into the nucleus.

Lack of persistence of DNA.
Lack of tissue targeting.
Some genetic diseases of gene therapy:
1. Cystic fibrosis:
CF is an autosomal recessive disorder. It may happen due to
abnormal electrolyte transport across the surface of epithelial cells.
This disease causes respiratory failure as a result of bacteria colonizing
the airways.
Causes:
Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) have been pinpointed as
the cause of the disease. CFTR is a cAMP-mediated chloride
channel.
Mutation in the protein prevent proper Cl channel opening,
hence inhibiting the movement of water across the epithelial
surface. As a result, the mucus within the airways becomes sticky
which ultimately enhance bacterial infection. This disorder also
causes blockage in the intestine and sterility.
Treatment:
Present treatment involves administering antibiotics and enzyme
supplements.
2. Adenosine deaminase deficiency:
Adenosine deaminase (ADA) deficiency is a rare autosomal
recessive disorder that results in severe combined immunodeficiency.
Causes:
This disease is caused by defective expression of the enzyme
ADA that plays a major role in the purine salvage pathway. Absence
of the enzyme results in elevated levels of dATP blocking Tlymphocyte differentiation in the thymus.
Treatment:
Current therapy includes bone marrow transplant.
3. Familial hypercholesterolemia:
FH is frequently occurring autosomal dominant disorder occurring
in 1:500 births.
Causes:
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FH results from the absence of low density lipoprotein (LDL)

receptors and as a consequence the level of LDL remains elevated
in the blood while high density lipoprotein (HDL) is suppressed. The
condition often results in premature development of atherosclerosis,
a condition in which the arteries become thickened and eventually
blocked. If this occurs in the major coronary arteries, the patient
can suffer angina and acute myocardial infarction.
Treatment:
At present, coronary heart disease is treated with bypass
operations, which involve the grafting of new arteries or coronary
angioplasty that involves the insertion of a tiny balloon into the
blocked artery which is then inflated to remove the obstruction.
4. Hemophilia-A
5. Hemophilia-B
6. Thalacemia
7. Sickle cell anaemia
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Biotechnology is the application of scientific and engineering principles

altered bacteria in the cleanup of oil spills.

products or processes for specific use."

when used to produce industrial goods.

engineered to express a pesticide, thereby eliminating the need for

external application of pesticides.

What is the difference between biotechnology and

development of drugs or therapies.

The therapeutic proteins and hormones, fermentation products like the

hybridoma technology, Mutagenesis etc.

1979: NIH guidelines relaxed. Genentech clones human growth

A-interferon and first recombinant vaccine

for hepatitis B was approved.

model, First patent for genetically altered animal.

Biotechnology Applications for world food production

identification of genotypes through DNA fingerprinting.

pathogens, with economic implications for pathogen monitoring and

Biotechnology: Biotechnology is the application of recombinant DNA

techniques in commercial enterprises. There are three main areas:

biomass by genetically improved yeast or bacteria.

used in laundry detergent.

2) Agriculture: Extensive use of biotechnology has been made to modify

express human insulin.

to replace damaged or diseased tissues in the body.

genetically modified food has been sold since 1994.

In short, genetic engineering is the technology of preparing

together fragments from more than one organism.

Recombining of DNA Technology

are of value to science, medicine, agriculture, or industry.

research or medicinal use, such as commercially produced insulin).

construct is built containing all the genetic elements for correct

and the presence of the new genetic material is tested for.

Essential steps involved in the expression of protein genes:

complementary RNA strand, which is called the primary transcript.

expression is complete after a functional tRNA or rRNA is generated.

However, protein genes require additional steps:

cytoplasm for protein synthesis.

The central dogma:

Steps involved in the preparation of recombinant DNA:

5. A host to replicate the vector containing foreign DNA. This may be

yeast artificial chromosome (YAC).

Table: Commonly used restriction enzymes:

Isolation of recombinants DNA clones:

Figure: The essential steps in DNA cloning using plasmids as

a) DNA recombination. The DNA fragment to be cloned is inserted into a

c) Selective amplification. A specific antibiotic is added to kill E. coli

DNA cloning using lamda phages as vectors:

Figure: Schematic drawing of the DNA cloning using l phages as vectors.

The DNA to be cloned is first inserted into the l DNA, replacing a

Polymerase Chain Reaction (PCR) :Primers are in green color:

temperature (> 60o C).

is gradually cooled to about 60o C.

Much faster than using vectors.

entire genome of an organism.

synthesized from mRNA molecules in a cell.

about 1000 times higher than the plasmid vector.

Figure: Preparation of the genomic library using l phage vectors. It is

Schematic diagram of recombinant DNA technology is given

Extraction of total RNA

Preparing cDNA by reverse transcription

using reverse transcriptase enzyme

Doing agarose gel electrophoresis to isolate