Polyclonal antibodies are produced by injecting an antigen into a host animal which causes its immune system to produce various antibodies that recognize different epitopes of the antigen. The antibodies are then purified from the animal's blood plasma. Polyclonal antibodies are a heterogeneous mixture that can bind to multiple epitopes of the same antigen, whereas monoclonal antibodies are all clones that recognize the same epitope.

1. Presented to: Madam Maliha
Antibodies, Polyclonal and Monoclonal antibodies

2. Table of Content
● What are antibodies
● Structure, function and types of antibodies
● Polyclonal antibodies
● Production of polyclonal antibodies
● Purification of polyclonal antibodies
● Function of polyclonal antibodies
● Advantages and disadvantages of polyclonal antibodies

3. ● Monoclonal antibodies
● Production of monoclonal antibodies
● Purification of monoclonal antibodies
● Function of monoclonal antibodies
● Advantages and disadvantages of monoclonal antibodies
● Differences between polyclonal and monoclonal antibodies
● Applications
● Key points
● References

4. Group members
● Ramaieta saleem
● Muazma nabi
● Usama Aamir
● Abdul basit

5. Introduction to Antibody
By : Ramaieta saleem (41405)

6. Antibody
● Antibody, also called immunoglobulin, a protective protein
produced by the immune system (B cells) in response to
the presence of a foreign substance (antigen).
● It recognize and latch on to the antigen in order to remove
them from the body.

8. Mechanism
● The most important mechanism to eliminate invader is the
production of antibody.
● Stimulation: As antigen binds to the B cells surface it
stimulates B cells to divide and mature in to group of
identical cells called clone.
● Mature B cells also called plasma cells which secrete
millions of antibodies into blood stream and lymphatic
system.

10. Function of antibody
● Antibody circulates, attack and neutralize the antigen
which activate the immune system.
● Antibodies attack antigens by binding to them.
● Neutralization:When antibody bind to a toxin it neutralize
poison by altering its chemical composition such
antibodies are called antitoxins. For example diphtheria
antitoxin.

11. ● By attaching with invader microbe it can make
microorganisms immobile or prevent them from
penetrating body cells.
● Opsonization: The coating antigen for more phagocytosis.
Activation of complement:
● Trigger the lysis( bursting ) of the invaders and attract
microbe killing scavenger cells that ingest or phagocytose
the invader.

12. Antigen receptors
● For the production of antibodies it is important that B cells
bind with antigen. Each B cells bind only one type of
antigen and it recognize countless number of antigens.
● B cells surface contain antigen specific receptors (antibody
protein) recognize antigen.
● Particular B cells have identical antigen receptors but on
other it differ.

13. ● Structural variation among antigen binding sites allows
different B cells to recognize different antigens.
● Due to binding between receptor and epitope(antigenic
determinant) that are complement to each other activates B
cells to produce antibodies.

14. Structure of antibody
● Antibodies are glycoproteins composed of
polypeptide chains and carbohydrate
● Monomeric structure Polypeptide chains
2 identical heavy chains
2 identical light chains
● Polypeptide chains joined by disulfide bonds
● Carbohydrate

16. LIGHT CHAIN:
• The light chain may be either of two distinct forms called
“Kappa” and “Lambda” and can be distinguished by AA
sequence of carboxyl portion of the chain.
HEAVY CHAIN:
• In the heavy chain NH2 terminal h has a pattern of variability
similar to that of kappa and lambda of the light chain.

17. Both light and heavy chains contain 2 different regions
constant and variable region.
• The four chains are arranged in the form of a flexible “Y”
with the hinge region and is termed as crystallizable fragment
(Fc) and contains the site at which Ab binds.
• Top of the “Y” consist of two Ag binding fragments (Fab)
that bind with antigenic determinant sites.

18. ● variable region
An antibody is made up of a variable region and a constant
region, and the region that changes to various structures
depending on differences in antigens is called the variable
region.
● constant region
The region that has a constant structure is called the constant
region.

19. ● Serum containing antigen-specific antibodies is called
antiserum.
● The 5 types – IgG, IgM, IgA, IgD, IgE – (isotypes) are
classified according to the type of heavy chain constant
region, and are distributed and function differently in the
body.
Classes of Antibody

21. IGg
● Immunity to new born.
● Potent activator of complement system.
● Neutralization of toxin.
● IgG3 binds to fc receptors phagocytosis.

22. Function of IgM
● Activation of classical pathway.
● Defence against multivalent bacterial antigens.
● Acts as opsonin.
● Present on B cells surface acts as antigen receptor

23. IgA
● Secretory antibody.
● Effective against bacterial like salmonella, vibriocholerae,
niesseria gonorrhoea, etc.
● Effective against viruses like those causing poliomyelitis
and influenza.
● Protection to infant gut.

24. Function of IgD
● B cell activation.
● Acts as receptor for Ag binding.
Function of IgE
● Responsible for immediate hypersensitivity or allergic
reactions.
● Binds to Fc receptors on basophils and mast cells.
● Release of substance like histamine, brady kinin and other
vasoactive mediators

25. Monoclonal and Polyclonal antibodies
● Monoclonal antibodies (mAb or moAb) are antibodies that
are made by identical immune cells that are all clones of a
unique parent cell. Monoclonal antibodies can have
monovalent affinity, in that they bind to the same epitope.
● Polyclonal antibodies (pAbs) are antibodies that are secreted
by different B cell lineages within the body (whereas
monoclonal antibodies come from a single cell lineage). They
are a collection of immunoglobulin molecules that react
against a specific antigen, each identifying a different epitope.

26. Polyclonal Antibodies
Presented by Muazma Nabi

27. What are Polyclonal antibodies:
● A polyclonal antibody is a collection of many
immunoglobulins each generated from a different B-cell
clone. These antibodies target different epitopes, or binding
sites, on a single antigen. OR,
● Polyclonal antibodies (pAbs) are heterogeneous mixture,
which are usually produced by different B cell clones in the
body. They can recognize and bind to many different
epitopes of single antigen.

28. ● Polyclonal antibodies refers to a mixture of
immunoglobulin molecules which are secreted against a
particular antigen.
● Each antibody recognizes different epitopes.
● This means polyclonal antibodies are a heterogenous
mixture of antibodies.

29. Polyclonal antibodies

30. Production of polyclonal antibodies:
● All antibody generation, polyclonal or monoclonal, begins
with eliciting an immune response, termed immunization.
● The intended targets are injected into the host, recognized
by the immune system as foreign, and targeted by
antibodies for immune blockage or clearance.
● Failure to mount an immune response would require the
use of adjuvants, immunogenic substances such as Freund's
adjuvant or keyhole limpet hemocyanin (KLH), to further

31. drive immune recruitment. Immunization comprises the
majority of what is required to make polyclonal antibodies.
General steps for production of Polyclonal antibodies:
● Polyclonal antibodies are often obtained by injecting a
lab animal most commonly rabbits, goats and mouse
with a specific antigen, to develop this antibody.
● We inject the antibodies inside the our organism by
inject.

32. ● After a fews weeks mostly, just after six weeks, the
immune system of animal will start producing the high
level of antibodies specific for the antigen.
● The plasma cells, present inside the body of that animal
starts producing antibodies.
● Then we extract these antibodies from the body of that
animal and purify all these antibodies.
● Normally, in this case the antibodies which are produce are
IgG along with variations.

33. Production of polyclonal antibodies

34. Initial Exposure for Prime Immune Response:
● Initial host exposure to antigen with, appropriate adjuvants,
exposure is termed "Prime" immunization.
● Typical methods of exposure include injection into the
skin, peritoneum, subcutaneous layer, or muscle. Less
common alternatives are oral and intranasal administration.

35. ● DNA immunization is also an emerging technology for
toxic or otherwise difficult to produce antigens. Any
subsequent exposure to antigen is termed a "boost"
immunization.
Antigen Processing by Antigen Presenting Cells:
● Macrophages, Dendritic Cells (DC), and to a lesser extent
B-Cells, all antigen presenting cells (APCs), internalize
antigen by endocytosis or phagocytosis.

36. ● Antigen processing breaks the antigen into smaller pieces
which are then moved to the surface of the APC.
Stimulation of B-Cells:
● Recognition of antigen presented on the surface of APCs
induces the release of stimulatory signaling molecules,
cytokines, from Helper T-Cells.
● These cytokines activate B-Cells which also recognized
antigen through their B-Cell receptor (BCR).

37. ● Some B-cells do not require Helper T-Cell activation after
BCR-antigen interaction, usually producing a quicker
immune response with lower affinity antibodies.
● The involvement of Helper T-Cells is critical in a replete
immune response as well as regulating self-tolerance
Proliferation of B-Cells and Antibody Secretion:
● Activated B-Cells divide to create both memory B-cell and
plasma cell populations. Both types of cells recognize the

38. Plasma cell development

39. same epitope.
● Plasma cells secrete antibodies into the serum for immune
recognition while memory B-Cells persist for longer
periods, providing quick response to secondary exposure of
the same antigen.
Purification of Polyclonal antibodies:
Purification process with a combination of chromatography

40. Steps and precipitation methods, one can purify antibodies
with good yield and resolution.
Why to purify Polyclonal antibodies:
● Basically they need to purify in order to remove all the
possible contaminants like albumin, transferrins and cell
degradation products like DNA and cellular proteins.

41. Purification Methods:
● The choice of purification methods is generally based on
the factors mention below;
1. Nature of antibody
2. Nature of feedstock.
3. Scale of production
4. Economic cost and other factors
5. Process timing and Desired purity

42. Process of purification:
● This process is basically multi-step process which mainly
include;
1. Sample preparation.
2. Capture.
3. Initial purification.
4. Secondary purification.
5. Polishing or formation.

43. Advantages of Polyclonal antibodies:
● Inexpensive to produce.
● Quick to produce. Purified antibody ready to use in
under four months.
● Easy to store.
● Highly stable and tolerant of pH or buffer changes.
● Higher overall antibody affinity against the antigen due
to recognition of multiple epitopes.

44. ● Superior antibody affinity generally results in quicker
binding to target antigen. Ideal in assays requiring quick
capture of the protein such as IP or ChIP.
● Significantly more robust when assaying proteins that
show slight variations in individual epitopes such as
denaturation, polymorphism or conformational changes.
● Superior for use in detecting a native protein in multiple
assay types.

45. ● Much easier to couple with antibody labels. Less likely to
affect binding capability.
Disadvantages:
● Variability between different batches produced in
different animals at different times
● Higher potential for cross reactivity due to recognizing
multiple epitopes
● Affinity purification of the serum will typically be
required to minimize cross reactivity

46. Uses of polyclonal antibodies
● Polyclonal antibodies are used experimentally and in
clinical medicine for a variety of reasons.
● Polyclonal preparations are generally easier and less
expensive to generate than monoclonal antibodies, and
they are also able to withstand greater variation in
temperature and pH.
● In medicine the most common use of polyclonal
antibodies is to confer passive immunity to certain disease.

47. ● The only effective treatment for Ebola, for example, is a
transfusion of serum antibodies from a human survivor of
the virus.
● In diseases such as Ebola this is effective because the virus
multiplies and acts so quickly within the body that the
immune system does not have time to mount its own
defense.
● When someone becomes infected with the Ebola virus, he
or she dies long before the immune system can combat the
virus.

48. ● The only effective treatment is therefore the protection
conferred by the antiserum of someone who has
encountered the infection previously.
● Another medical use for antiserum is as antitoxin or
antivenin.
● These preparations contain antibodies specific for venom
from poisonous reptiles, arachnids, and insects.
● They are used to treat people who have been bitten or stung
by these animals, again because the venom acts too quickly
in the body for the immune system to mount a defense.

49. Monoclonal Antibodies
By : Usama Aamir (41585)

50. The idea of "magic bullets" was first
proposed by Paul Ehrlich, who, at
the beginning of the 20th century,
postulated that, “ If a compound
could be made that selectively
targeted a disease-causing
organism, then a toxin for that
organism could be delivered along
with the agent of selectivity.”
History And Discovery

52. What are Monoclonal Antibodies
● Monoclonal antibodies are identical immunoglobulins,
generated from a single B-cell clone.
● These antibodies recognize unique epitopes, or binding sites, on
a single antigen.
● Derivation from a single B-cell clones and subsequent targeting
of a single epitope is what differentiates monoclonal antibodies
from polyclonal antibodies.

53. ● Therapeutic monoclonal antibodies act through multiple
mechanisms, such as blocking of targeted molecule functions,
inducing apoptosis in cells which express the target, or by
modulating signalling pathways
● Antibodies can also be used to purify their target compounds from
mixtures, using the method of immunoprecipitation.

54. Production of Monoclonal Antibodies
● We use hybridoma technology to produce large amount of
Monoclonal Antibodies.
● Hybridoma technology is a method for producing large numbers of
identical antibodies or Monoclonal Antibodies.

64. Nomenclature
Suffix - use at the end of Antibody name.
● Human : umab
● Humanized : zumab
● Murine : momab
● Chimeric : ximob

66. Diagnostic Applications
● MAbs in Biochemical Analysis
● Diagnostic tests based on the use of MAbs as reagents are routinely
used in radioimmunoassay (RIA) and enzyme-linked
immunosorbent assays (ELISA) in the laboratory.
● These assays measure the circulating concentrations of hormones.
example : insulin, human chorionic gonadotropin.

67. ● it is now possible to do the early diagnosis of the following
conditions/diseases through MAb’s.
● Pregnancy.
● Cancers.
● Hormonal disorders.
● Infectious diseases.
● Monoclonal antibodies are successfully used in the diagnostic
imaging of cardiovascular diseases, cancers and sites of bacterial
infections.

68. Therapeutic Applications
MAbs are used in the treatment of cancer, transplantation of bone
marrow and organs, autoimmune diseases, cardiovascular diseases and
infectious diseases.
The therapeutic applications of MAbs are broadly grouped into 2
types:
(A) Direct use of MAbs as therapeutic agents
(B) MAbs as targeting agents.

69. (A) MAbs as Direct Therapeutic Agents
● In destroying disease-causing organisms.
● In the treatment of cancer.
● In the immunosuppression of organ transplantation.
● In the treatment of AIDS.
● In the treatment of autoimmune diseases.
(B) MAbs as Targeting Agents in Therapy
● MAbs in use as immunotoxins.
● MAbs in drug delivery.
● MAbs in the dissolution of blood clots.
● MAbs in radio immunotherapy (RAIT).

70. Side effects
● Allergic reactions, such as hives or itching.
● Flu-like signs and symptoms, including chills, fatigue, fever, and
muscle aches and pains.
● Nausea, vomiting.
● Diarrhea.
● Skin rashes.
● Low blood pressure.

71. Growth of Monoclonal Antibody Market
Vast growing number of approved product in the market will
increase the incidence of monoclonal market in the coming future.
Current Scenario

72. Summarization
By : Abdul Basit (42512)

73. Advantages and Disadvantages of Monoclonal Antibodies
Advantages Disadvantages
● Highly specific recognition of only
one epitope of an antigen.
● Immortal hybridoma cell lines have
the ability to produce unlimited
quantities of antibodies.
● High consistency among experiments
● Minimal background noise and
cross-reactivity.
● Excellent for affinity purification.
● Developing a monoclonal takes time
and requires high technical skills.
● They can produce large amounts of
specific antibodies but may be too
specific to detect in across a range of
species.
● Vulnerable to the change of epitope.
Even a slight change in conformation
may lead to dramatically reduced
binding capacity.

74. Advantages and Disadvantages of Polyclonal Antibodies
Advantages Disadvantages
● Short production time and low cost.
● Highly stable and tolerant of pH or
buffer changes.
● High affinity. Since the antibodies
bind to more than one epitope, they
can help amplify the signal from
target protein even with low
expression level. This makes these
antibodies ideal for
immunoprecipitation and chromatin
immunoprecipitation.
● Prone to batch to batch variability.
● Multiple epitopes make it important
to check immunogen sequence for
any cross-reactivity.

75. Advantages of Polyclonal Antibodies
● Tolerant of minor changes of antigen. Polyclonal antibodies
are less sensitive to antigen changes (slight denaturation,
polymorphism, heterogeneity of glycosylation) than
monoclonal antibodies.
● They're inexpensive to make and can be generated fairly
quickly, taking up to several months to produce.

77. KEY CONCEPTS
● Antibodies bind with high specificity to antigens used to
challenge the immune system, but they may also show cross-
reactivity by binding to other antigens that share chemical
properties with the original antigen.
● Injection of an antigen into an animal will result in a
polyclonal antibody response in which different antibodies are
produced that react with the various epitopes on the antigen.

78. ● Polyclonal antisera are useful for some types of laboratory
assays, but other assays require more specificity. Diagnostic
tests that use polyclonal antisera are typically only used for
screening because of the possibility of false-positive and false-
negative results.
● Monoclonal antibodies provide higher specificity than
polyclonal antisera because they bind to a single epitope and
usually have high affinity.

79. ● Monoclonal antibodies are typically produced by culturing
antibody-secreting hybridomas derived from mice. mAbs are
currently used to treat cancer, but their exorbitant cost has
prevented them from being used more widely to treat
infectious diseases. Still, their potential for laboratory and
clinical use is driving the development of new, cost-effective
solutions such as plant bodies.

80. References
● Amyx HL . 1987 . Control of animal pain and distress in antibody production and
infectious disease studies . JAVMA 191 : 1287 – 1289 .
● Anon . 1989 . Code of Practice for the Production of Monoclonal Antibodies .
Rijswijk, The Netherlands : Veterinary Public Health Inspectorate .
● AVMA [American Veterinary Medicine Association] . 2000 . Report of the
AVMA Panel on Euthanasia . JAVMA 218 : 669 – 696 .
● Borrebaeck CA Danielsson L Moller SA . 1988 . Human monoclonal antibodies
produced by primary in vitro immunization of peripheral blood lymphocytes .
Proc Natl Acad Sci U S A 85 : 3995 – 3999 .

81. References
● Brodeur BR Tsang PS . 1986 . High yield monoclonal antibody production in
ascites . J Immunol Methods 861 : 239 – 241 .
● BVA/FRAME/RSPCA/UFAW [British Veterinary Association/Fund for the
Replacement of Animals in Medical Experiments/Royal Society for the Prevention
of Cruelty to Animals/Universities Federation for Animal Welfare] . 1993 . Joint
working group on refinement: Removal of blood from laboratory mammals and
birds . Lab Anim 27 : 1 – 22 .
● Cox JC Coulter AR . 1997 . Adjuvants—A classification and review of the modes
of action . Vaccine 15 : 248 – 256 .
● Griffen PS Turton J Andrews CM Barrett P Clarke CJ Fung KW Munday MR
Roman IF Smyth R Walshe K York MJ . 2003 . Markers of experimental acute
inflammation in the wistar han rat with particular reference to haptoglobin and C-
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