immunology

1. Mr. Bulet Kumar Gupta
Assistant Professor,
Sai College of Pharmacy, Mau
Immunology

2. Immunology
Immunity is your body's ability to recognize germs to prevent them from
causing illness. The immune system's job is to help identify and eliminate
dangerous germs that enter the body before they can cause disease or
damage. There are two types of immunity: innate and adaptive.
 Innate Immunity
Innate immunity is the immune system that is present when you are born.
It is your body's first line of defense against germs. It includes physical
barriers, such as skin and mucous membranes, and special cells and
proteins that can recognize and kill germs. The problem with these special
cells and proteins is that they can kill a germ, but once the germ is dead,
the innate immune system forgets it. It does not communicate any
information about the germ to the rest of the body. Without this
information, the body cannot prepare itself to fight this germ if it should
reinfect the body.
What is Immunity?
Immunoglobulins in blood

3.  Adaptive Immunity
Adaptive immunity is protection that your body builds when it meets and remembers antigens, which is another
name for germs and other foreign substances in the body. When your body recognizes antigens, it produces
antibodies to fight the antigens. It takes about 14 days for your body to make antibodies. More importantly, the
body memorizes this fight so that if its meets the same antigen again, it can recognize and attack more quickly.
Antibody production is one of the most important ways that immunity is developed. There are two types of adaptive
immunity: active and passive.
•Active Immunity - antibodies that develop in a person's own immune system after the body is exposed to an
antigen through a disease(natural) or when you get an immunization(artificial or vaccine induced immunity)
(i.e. a flu shot). This type of immunity lasts for a long time.
•Passive Immunity - antibodies given to a person to prevent disease or to treat disease after the body is exposed
to an antigen. Passive immunity is given from mother to child through the placenta before birth, and through
breast milk after birth. It can also be given medically through blood products that contain antibodies, such as
immune globulin. This type of immunity is fast acting but lasts only a few weeks or months.
Immunology

4. Active immunity is the immunity which get stimulated by exposure to antigens. It is mediated by two mechanisms:
•Cell-mediated Immunity
•Humoral Immunity.
Both immune pathways differ in their targets, components and methods of destroying pathogens.
Both comes under adaptive immunity
Humoral Immunity(B-cells )
Humoral immunity is mediated by antibodies. It shows a quick response against pathogens. It is the major defence
mechanism against extracellular microbes trying to invade the host systems. The antibodies produced by the B-
cells bind to the antigens and neutralize the microbes. foreign material typically includes extracellular invaders such
as bacteria are detected by this. T-cell independent immunity.
Cell-Mediated Immunity(T-cells)
Cell-mediated immunity is facilitated by the T-helper and cytotoxic T-cells. Cytokines secreted by the T- helper cells
activate phagocytic cells, which phagocytose the pathogens and kill them. T-cell dependent immunity.
Immunology

5. Similarities between Cell-mediated and
Humoral Immunity
•Both humoral and cell-mediated immunity
are active immunities.
•Both have a lag period.
•Both are active against a wide variety of
pathogens.
•Both possess immunological memories.
•Both systems are not effective in immune-
deficient individuals.
Difference and similarity between cell-mediated and humoral immunity

6. Immunology

7. Naïve B cells are lymphocytes that circulate throughout the body in the lymphatic system. These lymphocytes
express a variety of antigen-specific molecules that are essential for the detection of infectious agents in the human
body. Whenever naïve B cells encounter an antigen in the lymphatic system, they undergo a differentiation process
that leads to the creation of memory B cells and effector B cells.
What is an antibody?
Antibodies are heavy proteins that are approximately 10 nanometers in size.
These molecules are produced by B cells in order to identify and neutralize
harmful agents such as infectious bacteria, fungi, and viruses. These Y-
shaped proteins contain antigen-binding sites that specifically bind to their
target antigens.
Once antibodies effectively bind to their target antigen, they can either
neutralize their target antigen directly by blocking normal antigen binding or
they can stimulate other immune cells or molecules that promote the
antigens removal or destruction.
Immunology
Immunoglobulins or antibodies in
blood

8. How are antibodies produced?
Each B cell produces its own set of antibodies with unique antigen-specific binding sites. Initially, naïve B cells
produce antibodies that remain bound to the cellular surface so that their exposed antigen-binding sites can detect
potential pathogens, toxins and foreign material. This surface-bound form of an antibody is known as an
immunoglobulin.
When an antigen matching the antigen-binding site binds to a naïve or memory B cell, it activates the B cell to
produce and secrete more antigen-specific antibodies. Once a B cell fully matures, it is known as a plasma cell
and will continue to produce and secrete antigen-specific antibodies for the remainder of its life cycle.
Immunology

9. Immunoglobulins
Immunoglobulins or antibodies are heterodimeric proteins composed of two heavy (H) and two light (L) chains.
They can be separated functionally into variable (V) domains that binds antigens and constant (C) domains that
specify effector functions such as activation of complement or binding to Fc receptors.
Classes of immunoglobulins
The five primary classes of immunoglobulins are IgG, IgM, IgA, IgD, and IgE. These are distinguished by the type of
heavy chain found in the molecule.
IgM
• IgM is the first antibody produced in response to a microbial
attack by B cells.
• It is the largest antibody and is found in a pentameric form.
• It circulates in the blood and lymph and constitutes 6% of the
total antibody content in the serum.
• It is involved in agglutination and opsonization.
• It has a large number of antigenic sites on its surface and
therefore facilitates efficient activation of the immune system.

10. IgG
• Most abundant isotype in the plasma, and
comprises 80% of the total antibody content in the
serum. It detoxifies substances that are harmful
and recognizes the antibody-antigen complex.
• It is transferred to the placenta through the foetus
and protects the infant until its birth.
• It facilitates the process of phagocytosis and
provides immunity to the developing foetus. It
neutralizes the toxins and pathogens and offers
protection to the body.
IgA
• Usually found in liquids such as breast milk, serum,
saliva, fluids of the intestine. IgA in breast milk protects
an infant’s gastrointestinal tract from microbial activity.
• It constitutes 13% of the total antibody content in the
serum highly found in the secretions and is also called
the secretory immunoglobulin.
• It exists in both monomeric as well as dimeric forms.
• It provides the first line of defence against the
pathogens and limits inflammation. It also activates the
complement pathway and participates in the immune
response.
IgD
• It is involved in the production of the antibody by B
cells.
• It is present as a monomer and comprises less than 1%
of the total antibody content in serum.
• It acts as a receptor on B cell surface and participates
in B cell activation and differentiation.
IgE
• It is present in the least amounts, around 0.02% of the
antibody content in the serum.
• These are present in the linings of the respiratory and
intestinal tracts and respond to allergic reactions.
• Monomeric shape

12. Major histocompatibility complex(MHC)
MHC molecules act as a cell surface vessels (markers) for holding and displaying fragments of antigen so that approaching T cells can engage with this molecular
complex via their T-cell receptors. There are two types of MHC molecules such as MHC-I and MHC-II. MHC-I is predominantly present on the surface of all
nucleated cells where as MHC-II is expressed by blood cells, lymphocytes and dendritic cell so that the helper T-cell can recognize these surface markers and
activate the B-cells for destruction of those infected cells. Both MHC-1 and MHC-II are present in every cell in the body but the difference is healthy cell will
show more expression to MHC-I and the infected cell will show more MHC-II in there surface. In this way immune system recognize the healthy and disease cells.
MHC Class I:
•Both nucleated cells, as well as platelets, express MHC class I molecules—in other words, all cells except red
blood cells. Killer T cells, also known as cytotoxic T lymphocytes, are presented with epitopes (CTLs). CD8
receptors, as well as T-cell receptors (TCRs), are expressed by CTLs.
•When a CTL's CD8 receptor binds to an MHC class I molecule, and the CTL's TCR matches the epitope inside the
MHC class I molecule, the cell is conditioned to die through apoptosis.
•As a result, MHC class I plays a role in mediating cellular immunity, which is a key mechanism for combating
intracellular pathogens like bacteria or viruses, which include bacterial L types, bacterial genus Rickettsia and the
bacterial genus Mycoplasma. HLA-A, HLA-B, and HLA-C molecules make up MHC class I in humans.

13. Major histocompatibility complex(MHC)
MHC Class II:
•MHC class II are being represented conditionally by any cell type, but it is most commonly found on "trained"
antigen-presenting cells such as macrophages, B cells, and, in particular, dendritic cells.
•An APC picks up an antigenic protein, processes it, and then restores a molecular fraction of it—the epitope—to
view on the APC's surface, which is bound to an MHC class II molecule.
•Immunologic structures such as T-cell receptors (TCRs) may identify the epitope on the surface of the cell. The
paratope is the molecular region that connects with the epitope.
Major Histocompatibility Complex Function
 MHC is a tissue-antigen that helps the immune system (specifically T cells) to recognise, bind to, and accept itself
(auto recognition).
 The MHC-peptide complex is essentially an auto-antigen/alloantigen complex. T cells should accept the auto-
antigen after binding but activate when exposed to the alloantigen. When this theory is violated, illness arises.
 Antigen Presentation: MHC molecules bind to T cell domain to activate T cells.

15. Major histocompatibility complex(MHC)

16. Major histocompatibility complex(MHC)
 Normally when a cell is got diseased or infected with virus then there is a change inside the cell so it cannot
interact properly with neighbor cells and display cell surface marker MHC-I so that T-helper cells will recognize
this surface markers and activate the B-lyphocytes for destruction of this cells.
 In case of bacterial infection, the microbes are attached on the surface of the cell. Macrophages engulf the
microbes and lysis them into fragments. And this fragments they display on surface by MHC-II so that T-helper
cell will recognize and activate B-lymphocytes for producing antibodies against that microbe and T-cytotoxic will
destroy the infected cell.

17. Immune cells act as police man in body to eliminate
the microbes and diseased cells

18. Hypersensitivity
Hypersensitivity (also called hypersensitivity reaction or intolerance) refers to undesirable reactions produced by
the normal immune system, including allergies and autoimmunity.
They are usually referred to as an over-reaction of the immune system and these reactions may be damaging and
uncomfortable.
Hypersensitivity reactions can be classified into four types.
Type I: IgE mediated immediate reaction
Type II: Antibody-mediated reaction (IgG or IgM antibodies)
Type III: Immune complex-mediated reaction
Type IV: Cytotoxic, cell-mediated, delayed hypersensitivity reaction
The first three types are considered immediate hypersensitivity reactions because they occur within 24 hours. The
fourth type is considered a delayed hypersensitivity reaction because it usually occurs more than 12 hours after
exposure to the allergen, with a maximal reaction time between 48 and 72 hours.

19. Hypersensitivity

20. Hypersensitivity

21. The aims of immune stimulation are:
(1) to help bacterial killing at the primary focus of infection
(2) to prevent the development of nosocomial infections
(3) to prevent the reactivation of dormant viruses.
Immunostimulants, also known as immunostimulators, are substances (drugs and nutrients) that
stimulate the immune system by inducing activation or increasing activity of any of its components.
One notable example is the granulocyte macrophage colony-stimulating factor.
Classification
There are two main categories of immunostimulants:
1.Specific immunostimulants provide antigenic specificity in immune response, such as vaccines or
any antigen.
2.Non-specific immunostimulants act irrespective of antigenic specificity to augment immune
response of other antigen or stimulate components of the immune system without antigenic
specificity, such as adjuvants and non-specific immunostimulators.
Immune stimulation

22. Examples of immunostimulants are
• Tulasi, curcumin, aswagandha, Ginger
vaccines like
•HIV vaccine.
•Provenge.
•Remune.
•Sipuleucel-T.
Immune stimulation

23. Immunosuppressants
Immunosuppressive agent
An agent that decreases the body’s immune responses. It reduces the body’s ability
to fight infections and other diseases, such as cancer. Immunosuppressive agents
may be used to keep a person from rejecting a bone marrow or organ transplant.
They are also used in the treatment of conditions marked by overactive immune
responses, such as autoimmune diseases and allergies.
Glucocorticoids
•Cyclosporine. Cyclosporine is an immune suppressive drug used in the
treatment of immune diseases and transplant rejection. ...
Tacrolimus, Sirolimus, Everolimus, Mycophenolate mofetil, Mizoribine,
Leflunomide, Azathioprine.

25. Types of Immunization
Passive Immunization
 Methods of acquisition include natural maternal antibodies, antitoxins, and immunoglobulins
 Protection transferred from another person or animal
 Provision of temporary immunity by the administration of preformed antibodies
 Pooled human IG or IGIV
 Specific immune globulin preparations
 antitoxins
Active Immunization
 Methods of acquisition include natural infection,vaccines (many types), and toxoids
 Relatively permanent Can occur naturally via transfer of maternal antibodies across placenta to fetus
 Injection with preformed antibodies
 Human or animal antibodies can be used
 Injection of animal Ab’s prevalent before vaccines
 Effects are only temporary

26. Passive immunity
• Natural maternal antibody
• Immune globulin - An antibody-containing solution derived from human blood, obtained by cold ethanol
fractionation
of large pools of plasma; available in intramuscular and intravenous preparations
• Humanized monoclonal antibody
• Antitoxin - An antibody derived from the serum of animals that have been stimulated with specific antigens.
Active immunity
• Natural infection
• Vaccines - A suspension of attenuated live or killed microorganisms, or antigenic portions of them, presented
to a potential host to induce immunity and prevent disease.
o Attenuated organisms
o Inactivated organisms
o Purified microbial macromolecules
o Cloned microbial antigens
o Expressed as recombinant protein
o As cloned DNA alone or in virus vectors
o Multivalent complexes
• Toxoid - A bacterial toxin that has been modified to be nontoxic but retains the capacity to stimulate the
formation of
antitoxin
Acquisition of Passive and Active Immunity

27. Can occur naturally via transfer of maternal antibodies across placenta to fetus
Injection with preformed antibodies
• Human or animal antibodies can be used
• Injection of animal Ab’s
prevalent before vaccines
Effects are only temporary
The Immune System and Passive Immunization
The transfer of antibodies will not trigger the immune system
There is NO presence of memory cells
Risks are included
• Recognition of the immunoglobulin epitope by self immunoglobluin paratopes
• Some individuals produce IgE molecules specific for passive antibody, leading to
mast cell degranulation
• Some individuals produce IgG or IgM molecules specific for passive antibody,
leading to hypersensitive reactions
Passive Immunization

28. Natural Infection with microorganism or artificial acquisition (vaccine)
Both stimulate the proliferation of T and B cells, resulting in the formation of effector and memory cells
The formation of memory cells is the basis for the relatively permanent effects of vaccinations
Active Immunization
History and Achievements of Vaccines
During the 15th century, an early form of smallpox vaccination was
practiced in China and other
parts of the world. Healthy people were intentionally infected with
substances from the pustules of
people suffering from smallpox, a technique called variolation. A mild
form of smallpox usually
resulted from this practice.
An English doctor, Edward Jenner, improved the variolation technique
to create the first vaccine
in 1796. Dr. Jenner had heard that dairymaids who had been infected with
cowpox, a disease
related to but milder than smallpox, were not susceptible to smallpox, and
decided to test the
idea. He performed the first vaccination on a boy with material taken
from lesions of cowpox.
In fact, the word vaccination comes from the Latin word for cow,
vacca.

29. Common misconception that activation of the
immune system results in protective immunity
Multiple factors affect decisions when making
vaccines
1. Activation of specific branch of immune system
2. Development of immunological memory
Types of Vaccines
Whole-Organism
• Attenuated Viral/Bacterial
• Inactivated Viral/Bacterial
Purified Macromolecules
• Polysaccharide
• Toxoid
• Recombinant Antigen
• Recombinant-Vector
Development of Vaccines

30. Many common vaccines used consist of inactivated or attenuated bacterial cells or viral particles
Includes attenuated and inactivated vaccines
Attenuated Viral or Bacterial Vaccines
Attenuation – to reduce in force, value, amount, or degree; weaken
• Achieved by growth under abnormal culture conditions
• Bacillus Calmette-Guerin (BCG)
• Act as a double edged sword, as they have distinct advantages and disadvantages.
Advantages of Attenuated Bacterial or Viral Vaccines
Advantages stem from their capacity for transient growth
Prolonged immune-system exposure
Single immunizations
Replication within host cells
Exception to the Rule...
Sabin Polio vaccine consists of 3 attenuated strains of poliovirus
Whole-Organism Vaccines

31. Disadvantages of Attenuated Bacterial or Viral Vaccines
MAJOR disadvantage is possible reversion
• ex: Rate of reversion of Sabin Polio vaccine is one case in 4 million doses
Presence of other viruses as contaminants
Unforeseen post vaccine complications
The Future of Attenuation...
Genetic engineering techniques provide new methods of attenuation
Herpes virus vaccine for pigs
Possible elimination of reversion?
Colonization of intestine results in immunity to all 3 strains
• Production of secretory IgA and induction of IgM and IgG
Result is the need for boosters
• Individual strains interfere with one another
First immunization - one strain predominates in growth
Second Immunization - immunity generated by previous immunization limits growth of previously
predominant strain
Third Immunization - same principle as second immunization

32. Methods of inactivation include heat or chemical agents
• End result.... Loss of replication ability
Difficult to inactivate due to potential for denaturation of epitopes
• Dependence on higher order levels of protein structure
Inactivated Viral or Bacterial Vaccines

33. Subunit vaccines
• Vaccines made from well defined components of microorganisms are called a subunit vaccine
Recombinant vaccines
• A subunit vaccine that is produced using recombinant techniques is called a recombinant
vaccine.
Newer vaccines – Still Experimental
• DNA vaccine
• Peptide vaccine
• Anti-idiotype vaccine
Advantages of DNA vaccines
Plasmids are easily manufactured in large amounts
DNA is very stable
DNA resists temperature extremes so storage and transport are straight forward
DNA sequence can be changed easily in the laboratory.
By using the plasmid in the vaccinee to code for antigen synthesis,
Other forms of Vaccines

34. Possible Problems
Potential integration of plasmid into host genome leading to insertional mutagenesis
Induction of autoimmune responses (e.g. pathogenic anti-DNA antibodies)
Induction of immunologic tolerance (e.g. where the expression of the antigen in the host may lead to specific
non-responsiveness to that antigen)
Adjuvants
Adjuvants are CRITICAL for the use of inactivated vaccines
Most widely used are aluminum salts (mainly hydroxide or phosphate)
Effects include liberation of antigen, chemoattraction, and inflammation
Mixtures of plasmids could be used that encode many protein fragments from a virus/viruses so
that a broad spectrum vaccine could be produced
The plasmid does not replicate and encodes only the proteins of interest
There is no protein component and so there will be no immune response against the vector itself
there is a CTL response

35. Temperature control: Maintaining vaccines within the manufacturer’s recommended storage temperature during
transport and storage until the point of administration
Why is the “temperature control” so important?
Efficacy depends on correct storage conditions +2°C to + 8°C
Compliance with Specific Product Characteristics and marketing authorisation
Assurance and confidence in a potent product
Ensuring maximum benefit from immunization
Effect of Temperature on Vaccines
Live vaccines
tolerate freezing
deteriorate rapidly after removal from freezer
Inactivated vaccines
damaged by exposure to freezing temperatures
tolerate short time out of refrigeration
Storage conditions of vaccines

36. Storage
Storage of vaccines outside recommended storage temperatures can lead to:
Deterioration in the vaccine and failure to produce a satisfactory level of immunity
– Heat speeds up decline in potency - ↓shelf life
– Freezing causes
• Increased reactogenicity & loss of potency
• can lead to hair line cracks in ampoules, vials or pre-filled syringes causing contamination of contents
Temperature Sensitivity
• Sensitive to Cold and Heat
Light Sensitivity
• Sensitive to strong light, sunlight, ultraviolet and fluorescent light (neon)
• All vaccines should be stored in their original packaging until they are administered Vaccine Stability
Storage conditions of vaccines

37. Receipt and Transport
Storage
Temperature monitoring
Use in vaccination sessions
Disposal and spillage
Disruption of the cold chain
Storage and Management of Vaccines
Receipt of Vaccines
Checked against order for discrepancies
– Have vaccines been stored between 2oC – 8oC ?
Inspect for leakage and damage
Signed for and refrigerated immediately
Record vaccine type, brand, quantity and batch
numbers (date and time)
Transport of Vaccines
Insulated validated cool boxes
Cool boxes
– Fridge packs
– Frozen packs
Spaces in cool box filled with insulating material
Vaccines should not be in direct contact with cool packs
Vaccines taken to schools or outside clinics must be transported so
that the cold chain is maintained using validated insulated cool boxes
Then transferred to a fridge if available or left in a validated cool box
Unused vaccine transported in a validated cool box for a morning or
afternoon session may be returned to the fridge with a note attached
to use first
Vaccines stored for 8 hours or more in a validated cool box should be
disposed of and not returned to the fridge

38. Storage of Vaccines
Within recommended storage temperatures between 2 to 8 ◦C
Refrigerator Specifications:
– Designed for storing medicines- Lockable
• Minimal opening to maintain constant temperature
• Ice build up reduces effectiveness
– No items other than medicines stored in fridge (e.g. food, drink, clinical specimens)
– Should not be over full
– Ensure can not be accidentally switched off
– must not be removed from packaging during storage
– Stocks stored tidily
– Not stored on shelves in fridge doors or bottom drawers
– Not stored next to freezing compartments
– Patients/Parents should not be requested to store vaccines in a domestic fridge.
– Fridges should be cleaned on a regular basis
– Emergency storage available if fridge fails

39. Temperature Monitoring
Fridges must have a reliable maximum/minimum thermometer (in addition to any integral thermometer)
– Calibrate annually to ensure correct functioning
Designated person responsible for vaccine storage and fridge monitoring
– Trained to read and record current temperature, maximum and minimum temperatures correctly
Readings should be taken daily
Keep record chart on or near the fridge
Retain records until next audit
If the recorded temperature goes outside the range, contact community services pharmacy and or the manufacturer’s for
advice
Disposal of Vaccines
In Health Centres the box will then be either collected by pharmacy
technicians or returned to community services pharmacy on secure transport
GP Practices should make arrangements for disposal of vaccine waste
through their waste contractor

40. Hybridoma technology is one of the most common methods used to produce monoclonal antibodies. In this process,
antibody-producing B lymphocytes are isolated from mice after immunizing the mice with specific antigen and are
fused with immortal myeloma cell lines to form hybrid cells, called hybridoma cell lines. These hybridoma cells are
cultured in a lab to produce monoclonal antibodies, against a specific antigen. This can be achieved by an in vivo or
an in vitro method. thus produced are of high purity and are highly sensitive and specific.
Preparation of monoclonal antibodies using hybridoma technology
1. Immunization
The first step involves injecting the laboratory animals like rabbits or mice with a selected antigen against which the
antibodies are raised through a series of injections over a period of several weeks to stimulate B cell differentiation
into plasma B cells and memory B cells. Once a sufficient number of antibodies are created in the animal serum
following a few weeks of immunization, the animal is sacrificed.
2. Isolation of B lymphocytes
Following sacrifice, the spleen is removed in aseptic conditions to isolate the activated B-cells. This procedure is
performed using density gradient centrifugation. The presence of antibodies in the Serum is identified using methods
like ELISA or flow cytometry. The serum contains the activated B lymphocytes (that produce antibodies).
Hybridoma technology

41. The activated B lymphocytes are then fused with myeloma cells.
3. Preparation of myeloma cell lines
Few weeks before the cell fusion, metastatic tumor cells are incubated in 8-azaguanine to get non-functional
hypoxanthine-guanine phosphoribosyltransferase (HGPRT) genes in the myeloma cells. Non-functional HGPRT can
stop the assembly of nucleotides from the salvage pathway and makes the metastatic tumor cells sensitive to HAT
media as the preferred method in hybridoma technology.
4. Cell fusion
Cell fusion is the process in which the activated B lymphocytes are fused with HAT-sensitive myeloma cells. This step
is performed by centrifugation of freshly obtained activated B-cells with HAT-sensitive myeloma cells in a fusion-
promoting media. Polyethylene glycol (PEG) is used in this procedure. PEG helps in the fusion of cells by promoting
the fusion of the plasma membrane of the myeloma cells with the plasma membrane of the antibody-producing cells,
thus giving rise to a cell with more than one nucleus, forming heterokaryon. Another method used for fusion is
electrofusion, in which cells are fused under the effect of an electric field. This method is more efficient than the
previous method.

42. 5. Hybridoma selection
In the PEG-containing media, cells are fused to form hybridoma cells but even the most efficient fusion method will
allow the formation of only about 1 to 2% of fused hybridoma cells. Furthermore, about 1 in 100 cells will be viable
hybrid cells. Therefore, there are a number of unfused cells within the media. This step allows the selection of the
fused cells from all the unfused cells. This is achieved by incubating the cell mixture followed by culturing for 10–
14 days in HAT media (a selection media). HAT medium contains hypoxanthine-aminopterin-thymidine.
Aminopterin present in HAT media blocks the power of cells to synthesize nucleotides by the de novo synthesis
pathway. Hypoxanthine and deoxythymidine allow cells with functional hypoxanthine-guanine
phosphoribosyltransferase (HGPRT) genes to survive through salvage pathways. Due to a limited life span,
unfused B cells perish within a few days. Unfused malignant neoplastic cells die as a result of the lack of the
hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene. The presence of aminopterin blocks their ability
to synthesize nucleotides through the de novo pathway. Therefore, the remaining viable cells left in the media are
the hybrid cells; these hybrid cells have the ability to grow and divide on HAT media because they have functional
HGPRT gene from the B lymphocytes, which make them HGPRT positive, and thus, they can grow in unlimited
concentration on HAT media.

43. 6. Screening of hybridoma cells
HAT-selection hybridoma cells are transferred to ELISA plates, where each well houses a single hybridoma cell. This
is achieved using the limiting dilution method [8]. The genes of the B cell lineage present in the hybridoma cells
produce a specific antibody with a specific epitope; this antibody is known as “monoclonal antibody.”
7. Cloning and propagation of hybridoma cell
Hybridomas producing desired antibodies are selected and are then transferred into large culture vessels or flasks;
the hybridoma cell lines are cultured using in vivo or in vitro methods. These hybridoma cells can be maintained and
preserved in the culture media for the production of monoclonal antibodies.
8. In vivo
The in vivo method uses mice for the production of monoclonal antibodies. Mice are injected intraperitoneally with 105
to 110 viable hybridoma cells. After a few weeks, the ascites fluid is collected from an anesthetized mouse. Ascites
fluids are contaminated with mouse immunoglobulins to some extent and isolation of monoclonal antibodies requires
purification If purity of the antibody is important, then this technique may be inconvenient.
9. In vitro
This is another method in which hybridoma cells are cultured in laboratory conditions. It involves growing the hybrid
cells in a culture media followed by isolation of monoclonal antibodies from the media.