This document discusses techniques for producing antibodies, including both polyclonal and monoclonal antibodies. It describes the hybridoma technique developed by Kohler and Milstein, which involves fusing B cells that produce antibodies with myeloma cells to create immortal cell lines that produce monoclonal antibodies targeting a single epitope. The document outlines the characteristics of immunogens and antibodies, comparing polyclonal versus monoclonal antibodies. It also discusses applications of monoclonal antibodies in research, diagnostics, and therapeutics.

1. Immunological Techniques
ANTIBODIES PRODUCTION

2. Production of Antibodies
•In vitro and in vivo systems
•Hybridoma technique
•Monoclonal antibody
•Polyclonal antibody
•Phage displayed antibody
•Isolation of antibodies
•Bioaffinity columns
•Lectin and Antigen based columns

3. • In 1975, Kohler and Milstein first fused
lymphocytes to produce a cell line which
was both immortal and a producer of
specific antibodies.
• The two scientists were awarded the
Nobel Prize for Medicine in 1984 for the
development of this "hybridoma"
• The value of hybridomas to the field was
not truly appreciated until about 1987,
when MAbs were regularly produced in
rodents for diagnostics.
Production of Antibodies

4. In a specific immune response, only those T and B cells that can bind to
the antigens of the pathogen are selected to participate in the response.
Clonal selection of lymphocytes during the specific immune response
An antigen with 2 epitopes
- red epitope, blue epitope
Mixture of T and B cells
with different antigen
specificities
Proliferation of cells
with receptors capable
of binding epitopes of
the antigen
Production of antibodies

5. Production of antibodies
• If an animal is immunized with a protein, a wide array of B
cells will be stimulated to produce anti-protein antibodies.
• Antibodies may be made to a number of different epitopes
of the protein.
• Even antibodies that bind to the same epitope may have
different antigen-binding sites and bind the epitope with
different affinity.
• The mixture of antibodies produced in response to an
antigen are referred to as polyclonal antibodies (they are
produced by many different clones of B cells).
• Polyclonal antibodies are heterogeneous populations of
antibodies that are produced by various cell lines (or clones)
of B lymphocytes in response to various epitopes of the
same antigen

6. Production of antibodies
• Production of antibodies with high titer and specificities
is partly dependent on the type of antigens, stimulants
such as type of adjuvants, the route of entry, and number
of injections.
• All procedures regarding immunization of animals such
as injection of immunogen and bleeding of animals
require skill, patience, and practical training.
• The care and maintenance of laboratory animals must
meet the guidelines of the Institutional Animal Care and
Use Committees (IACUC).
• Immunogens must have a few characteristics, such as
foreignness, high molecular weight, chemical
complexity, and solubility

7. Production of antibodies
• Characteristics of Immunogens
– Solubility
• In order to produce an antibody, an antigen must be
immunogenic.
• Immunogen differs from antigen in a way that the former can
trigger an immune response and interact with the sensitized
cells and antibody produced, while the latter can bind with the
antibodies, but is not capable of producing an immune response.
• So, all immunogens are antigen, but not all antigens are
immunogens.
• A good immunogen contains three intrinsic characteristics:
– It must have an epitope that is recognizable by the B cell
surface antibody molecule.
– After processing, the degraded immunogen must offer at
least one site that can be recognized simultaneously by an
MHC class II protein and by a helper T cell receptor.
– Immunogen must be degradable.

8. Production of antibodies
• Characteristics of Immunogens
– Molecular Weight
– Large Molecules
•Large molecules, because of their higher degree of
conformation and structural rigidity, usually produce a
stronger immunogenic response.
•Polysaccharides, because of their complex
carbohydrate structures, are usually very good
immunogens.
•Various homopolymers of amino acids, although they
are large molecules, are not sufficiently chemically
complex, and so they are not good immunogens.
•Lipids usually are not immunogenic, but can be
immunogenic if they are conjugated to carrier protein.

9. Production of antibodies
• Characteristics of Immunogens
– Molecular Weight
– Small Molecules (Haptens) Coupled to a Carrier
Protein
•Haptens are small molecule antigens that can bind
to an antibody, but cannot elicit an adaptive
immune response.
•Haptens must be chemically linked to protein
carriers to initiate an immune response.
•So, a hapten is antigenic, but not immunogenic by
itself. Since an immunogen must have an epitope
or antigenic site and a class II T cell receptor
binding site, there is a minimum size necessary for
a molecule to be an immunogen.

10. Production of antibodies
• Polyclonal antibodies
– Polyclonal antibodies are a mixture of antibodies with
different antigen binding sites that may bind to
different epitopes or antigens of the immunizing
agent with varying affinities.
– They may be of different antibody classes.
– The serum obtained from an immunized animal is
referred to as a polyclonal antiserum.
– A polyclonal antiserum contains antibody to different
epitopes and different antigens that were present in
the immunizing inoculum

11. Epitopes
Immune Response
Antibodies
A mixture of antibodies - all bind to epitopes of
the original antigen. Some bind with higher
affinity than others.
Polyclonal antibodies
Protein
Immunize
Production of antibodies

12. • Monoclonal antibodies:
• Antibodies produced from a single clone of
B cells.
• Produced by fusing a B cell secreting the
desired antibody with a myeloma cell
capable of growing indefinitely in tissue
culture.
• Monoclonal antibodies all have identical
antigen-binding sites.
• Thus they all bind to the same epitope with
the same affinity.
• They are all of the same antibody class
(isotype).
Production of antibodies

13. Polyclonal antibodies
(Polyclonal antiserum)
B B B B B B B B
Harvest Ab
Monoclonal antibodies
Production of antibodies

14. Production of antibodies
• Poly- and Mono- Clonal Antibodies
– Polyclonal antibody
•Antigens possess multiple epitopes
•Serum antibodies are heterogeneous,
– To increase immune protection in vivo
– To reduces the efficacy of antiserum for various in
vitro uses
•To response facilitates the localization, phagocytosis, and
complement-mediated lysis of antigen
•To have clear advantages for the organism in vivo
– Monoclonal antibody
•Derived from a single clone, specific for a single epitope
•For most research, diagnostic, and therapeutic purposes

15. Production of antibodies

16. :Production of antibodies
Property Polyclonal antibodies Monoclonal Antibodies
Produced by: Many B cell clones A single B cell clone
Bind to: Multiple epitopes of all,
antigens used in the
immunization
A single epitope of a
single antigen
Antibody class: A mixture of different
Ab classes (isotypes)
All of a single Ab class
Ag-binding sites A mixture of Abs with
different antigen-
binding sites
All Abs have the same
antigen binding site
Potential for cross-
reactivity:
High Low

17. Antibody responses are generally polyclonal:
Many individual ‘clones’ make antibodies of varying affinities
against multiple epitopes found on the immunogen.
A polyclonal response is the sum of all of the clonal
responses (or monoclonal responses)

18. The selection of hybridoma cells is accomplished by culturing the fused cell mixture in
hypoxanthine-aminopterin-thymidine (HAT) medium. Aminopterin blocks the De Novo biosynthesis
of thymine, purines and pyrimidines. Myeloma cells do not survive because they are incapable of
growing when the De Novo nucleotide synthesis is blocked with HAT because they lack functional
hypoxanthine-guanine phosphoribosyl-transferase (HGPRT-). B cells die in vitro within two weeks.
The resulting hybridoma cells survive because they have received hypoxanthine-guanine
phosphoribosyl transferase (HGPRT+)from the B cells and immortality from the myeloma cells.
Monoclonal Antibody Production

19. Monoclonal v. Polyclonal Antibodies
Monoclonal: Limitless supply, defined specificity and subclass,
typically less ‘background reactivity’ and
less expensive, but sometimes application is limited
because only 1 epitope is recognized.
Polyclonal: Initially easier to produce, greater reactivity with
antigen (usually more applications), but not a
limitless supply. Specificity is less well-defined and
background reactivity is more of problem

20. • Humanized Monoclonal Antibodies
– Mouse monoclonal antibodies have been
genetically engineered to replace all of the
antibody molecule with human
counterparts except the hypervariable
regions directly involved with antigen
binding.
– Humanized monoclonal antibodies are
currently be tested in human clinical trials.
Production of antibodies

21. Production of antibodies
• Humanized Monoclonal Antibodies
– Production of human monoclonal antibody
•There are numbers of technical difficulties
– The lack of human myeloma cells to exhibit immortal
growth, be susceptible to HAT selection, to not secrete
antibody, and support antibody production in the
hybridoma made with them
– Human B cell sometimes have immortality
– That is the difficulty of readily obtaining antigen-
activated B cells
– To culture human B cells in vitro to produce human
monoclonal antibody
•Transplant human cells with immune response into SCID
mice (lack a functional immune system)

22. • Clinical Uses for Monoclonal Antibodies
• Measuring protein and drug levels in serum
• Typing tissue and blood
• Identifying infectious agents
• Identifying clusters of differentiation for the
classification and follow-up therapy of leukemias
and lymphomas
• Identifying tumor metastasis
• Identifying and quantifying hormones
• Immunoaffinity Purification
Production of antibodies

23. Production of antibodies
• Clinical Uses for Monoclonal Antibodies
– Very useful as diagnostic, imaging, and therapeutic
reagents in clinical medicine
•Monoclonal antibodies were used primarily as in
vitro diagnostic reagents
•Radiolabeled monoclonal antibodies can also be used
in vivo detecting or locating
– Immunotoxins
•To compose of tumor-specific monoclonal antibodies
coupled to lethal toxin
•Valuable therapeutic reagent

25. Potential Future Uses
• The remarkable specificity of antibodies makes
them promising agents for human therapy.
• Imagine, for example, being able to make an
antibody that will bind only to the cancer cells in
a patient coupling a cytotoxic agent (e.g. a strong
radioactive isotope) to that antibody, and then
giving the complex to the patient so it can seek
out and destroy the cancer cells (and no normal
cells).
Production of antibodies

26. Practical steps in monoclonal antibody production
1.Immunize animal
2.Isolate spleen cells (containing antibody-producing B cells)
3.Fuse spleen cells with myeloma cells (e.g. using PEG -
polyethylene glycol)
4.Allow unfused B cells to die
5.Add aminopterin to culture to kill unfused myeloma cells
6.Clone remaining cells (place 1 cell/well and allow each cell
to grow into a clone of cells)
7.Screen supernatant of each clone for presence of the desired
antibody.
8.Grow the chosen clone of cells in tissue culture indefinitely.
9.Harvest antibody from the culture supernatant.
10.(If you’re a biotech company) charge about $1,000-$2,000
per mg.
Production of antibodies

27. Kuby Figure 4-22

28. - B lymphocytes can mutate into tumor
cells that result in a type of cancer
termed myeloma.
- Myeloma cells become “immortal”
and will grow indefinitely in culture.
- Fusion of a single activated B cell and
a myeloma cell will create a
hybridoma that can grow indefinitely
in culture.
Hybridomas Technique

29. Harvest Ab
Monoclonal antibodies
Myeloma cells
Grow indefinitely in
cell culture but don't
secrete the desired
antibody
FUSE Hybridoma cells
Secrete antibody but don't
grow in tissue culture
Grow indefinitely in
cell culture AND
secrete antibody

34. Formation and Selection of Hybrid Cells
• Hybridoma: the B cell X myeloma cell
– To be produce by using polyethylene
glycol (PEG) to fuse cells
– The myeloma cells: immortal growth
properties
– The B cells: to contribute the genetic
information for synthesis of specific
antibody
– Selected by using HAT medium
(hypoxanthine, aminoprotein, and
thymidine)
•Myeloma cells are unable to grow
•B cells are able to survive, but can not
live for extended periods

35. Two different pathways to synthesis nucleotide in mammalian cells
(Folic acid analog)
Myeloma cells used in
hybridoma technology are
double mutants, they lack
the HGPRTase and lose the
ability to produce Ig
Formation and Selection of Hybrid Cells

36. (Most common screening
techniques are ELISA and RIA)
Low concentration
(1-20 ug/ml)
High concentration
(1-10 mg/ml)
Formation and Selection of Hybrid Cells

37. Formation and Selection of Hybrid Cells

38. • Myeloma cells have been genetically
engineered such that they can not use
hypoxanthine, aminopterin, and thymidine
(HAT medium) as a source for nucleic acid
biosynthesis and will die in culture.
• Only B cells that have fused with the
engineered myeloma cells will survive in
culture when grown in HAT medium.
Hybridoma Selection The “HAT Trick”

39. Hybridoma Selection The “HAT Trick”

40. Hybridoma Selection The “HAT Trick”
• The hybridomas are selected on a growth medium
containing hypoxanthine, aminopterin, and thymidine
(HAT).
• Aminopterin, which is a folic acid antagonist, blocks
the normal biosynthetic pathways for guanosine
production.
• When the normal biosynthetic pathway for guanosine
is blocked by aminopterin, there is an alternative
"salvage" pathway in which the nucleotide metabolites
hypoxanthine or guanine are converted to guanosine
monophosphate by the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT).

41. Hybridoma Selection The “HAT Trick”
• Cells lacking HGPRT (HGPRT－ cells) die in a HAT medium
because both the main and salvage pathways are blocked.
However, an HGPRT－ cell can grow in a HAT medium if it is
provided the missing enzyme by fusion with an HGPRT cell.
• The B lymphocytes provide the HGPRT enzyme to HGPRT－
myeloma cells.
• Heterokaryons are produced from binucleate fusion in the
presence of polyethylene glycol (PEG). The two nuclei fusion at
the next division, generating hybrid cells.
• Hybrid cells grow in a HAT medium, but both unfused spleen
and myeloma cells die.
• To select HGPRT－ cells, a toxic base analogue such as 6-
thioguanine or 8-azaguanine is used in the medium. HGPRT+
cells will incorporate a toxic base into their DNA and will die,
but HGPRT－ cells will survive.

42. Affinity chromatography:
1. Bind antibody to a support matrix (e.g.
sepharose gel)
2. Add protein mixture - antigen binds to
antibody on support
3. Wash to remove unbound material
4. Lower pH - antibody releases the antigen -
which is now free of contaminants
Affinity chromatography - protein
elution profile

43. From http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AffinityChrom.html
Affinity chromatography - antibody
purification.
Antigen can be bound to the support
matrix in order to purify antigen-specific
antibody from a polyclonal antiserum.
Engineering to create “immunotoxins”

44. Figure 6.21
Genetic engineering methods can be used to
modify monoclonal antibodies e.g. creation of
“humanized” monoclonal antibodies.
Antibodies of single specificity
(monoclonal) can be obtained by cloning
the appropriate heavy and light chain
genes.

45. Phage Display - Introduction
• The display of functional foreign
peptides or small proteins on the
surface of bacteriophage particles.
• As an important tool in protein
engineering
• As a powerful way to screen and select
for peptides on the basis of binding or
molecular recognition.

46. • More efficiently than through conventional hybridoma system.
• Cheaper to produce recombinant antibodies using bacteria,
rather than mammalian cell line.
• Easier to maintain and grow bacterial cultures for recombinant
antibody production.
• Bypass immunization in antibody selection.
• Bypass the use of animal cells for production of antibodies.
• Producing the combinatorial library (ideally with 108 to 109
members) of functional antibodies to generate a larger
repertoire of antibodies than those available through
conventional hybridoma technology.
• Easy isolation and expression of the cloned gene in a bacterial
host.
• Excellent potential to further improve binding properties of
the selected antibody by protein engineering techniques.
Phage Display - Advantages

47. Filamentous Phage
• To infect Gram-negative bacteria
• To adsorb specifically to the tip of F pili on
male cells.
• Be able to accommodate foreign DNA
fragments.
• Its non-lysogenic characteristic to permits
the extrusion of recombinant phage into the
culture supernatant.
• Long, thin, and flexible particles
that contain a closed circular
single-strained DNA molecule,
such as fd, f1, and M13.
• The major coat protein is pVIII.
The minor coat proteins pIII and
pVI are located at one end of the
phage; pVII and pIX are located
at the other end of the phage.

48. Filamentous Bacteriophage Vector
• To construct filamentous bacteriophage
vectors enabled the ‘‘biological’’
generation of the hundreds of millions
of unique peptides.
• Be the double-stranded replicative
form from a culture of inserted host cell.
• Be inserted an antibiotic resistance
gene.
• Be introduced a pair of specifically
situated endonuclease restriction sites
to allow cloning of DNA insert into a
position to express a foreign fusion
protein with capsid protein.

49. Phage Display Library
• Antibody library
 The sources of genetic material

50. Phage Display Library
Ways of construction

51. • Random peptide library
 The phage vector with which the library is
produced,
 The phage capsid component displaying
the peptide,
 The length of the insertion sequence,
 The choice of invariant residues that flank
the random sequence.
Phage Display Library

52. Flowchart of Phage Display Application
Displaying Targets on
Filamentous Phage
Panning (Selection) of
The Phage Library
Expression the Protein Fragment
or Isolation of Affinity-Purified
Phage Clones
Library clones
Expression
Isolation

53. Phage Display in Making Antibody
Generation of antibodies by immune system and phage display
technology
1: Rearrangement od assembly of germline V
genes
2: Surface display of antibody (fragment)
3: Antigen-driven or affinity selection
4: Affinity maturation
5: Production of soluble antibody (fragment)

55. The Plant body Approach

56. Screening Panels of Monoclonal Antibodies
Using Phage-Displayed Antigen
Miniplasminogen was displayed
on M13-type phage by fusion to
the NH2-terminus of the minor
coat proteinIII.

57. Structures of Binding Peptides Isolated from
Phage-Displayed Peptide Libraries
A turn-helix conformation is adopted
by a peptide that binds to the insulin-
like growth factor binding protein 1
(1998 in Biochemisty)
- from Current Opinion in Biotechnology (2000), 11, 610–616
A peptide that binds to the IgG-Fc
is a b-hairpin (2000 in Science)

58. Biosynthetic Phage Display
- from Chemistry & Biology (2000), 7, 263–274
a novel protein engineering tool combining chemical and
genetic diversity

59. Modification of Technique
• Panning of a Phage VH Library Using
Nitrocellulose
• Developments in the use of baculoviruses for
surface display
Development of phage display application
• Applying Phage Antibodies to Proteomics
• Applying phage display technology in aging
research

60. Purification of Antibodies
• Separation of the Ig fraction from a
mixture of antibody serum can be
achieved following salt fractionation and
chromatography procedures of protein
purification.
• Fractionation of serum proteins with
ammonium sulfate is a simple and
inexpensive procedure to enrich IgG
fraction.

61. Application of antibodies
• Specificity of Antibodies Make them Ideal
Reagents for Many Applications
– Therapy – Treatment against infection or
intoxication
– Diagnostics – Appearance of pathogen-
specific antibodies in serum is indicative of
exposure/infection
– Research Reagents – Molecular probes for
(protein) expression
– Chimeric and anti-Idiotypic antibodies.
– Isolation and purification of proteins

62. Therapy

63. A 20 year old diabetic college student returning from Spring
Break was concerned that he became infected with HIV.
How does his clinician help him?
Examine his serum by
ELISA for the resence
of HIV-specific
antibodies as a
marker for infection
Diagnostics

64. Research: Immunohistochemistry
Specific staining of pancreatic cells in murine
pancreas using low-power antigen-retrieval
(LAR) protocol. Insulin+ ß cells are shown in
green and pancytokeratin (CK+) pancreatic duct
epithelial cells in red (A1). Immunohistochemical
(IHC) staining of insulin was exclusively specific
for ß cells but not for duct epithelial cells (A2).
Insulin+ ß cells are the dominant cell population
in the islet (green), and cells are scattered
around the periphery of the islet (red) (B1). Low
magnification showing the highly specific
staining in the large area of tissue (B2); pancreatic
ß cells are stained with insulin in cytoplasm
(green) and PDX-1 in nuclei (red) (C1). There was
no unspecific binding in exocrine area (C2) and
specific staining of pancreatic duct with CK (red)
and vascular endothelial cells with PECAM
(CD31) (green) (D1). Ab against PECAM stained
only vessel but not duct (D2). Bars: A–C,D2 = 50
µm; D1 = 25 µm.