Lecture-12-Hybridoma-technique.ppt class

Technique for food analysis

1. Hybridoma TechniqueHybridoma Technique

2. 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.

3. 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

4. Polyclonal 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).

5. 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
B
B
B
B

6. 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.

7. 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.

8. B
B
B
B
Polyclonal antibodies
(Polyclonal antiserum)
B
B B B B B B B B
Harvest Ab
Monoclonal antibodies

9. - 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

10. B
Harvest Ab
Monoclonal antibodies
M
M
M
M
M
M
M
M
M
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

11. 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).

17. Inverted microscope

19. 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”

21. 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.

22. Kuby Figure 4-22

24. Rasing ascitic fluid

25. In vitro cultivation

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

27. 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
Uses

28. 99m
Tc-mAb
in vitro in vivo
Tumor diagnosis

29. Figure 6.24
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

30. From http://ntri.tamuk.edu/fplc/pursammat.html
Affintity chromatography - protein elution profile

31. 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).

32. 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.
Humanized Monoclonal Antibodies

33. Figure 6.21
Genetic engineering methods can be used to modify monoclonal antibodies
e.g. creation of “humanized” monoclonal antibodies.

34. Figure 6.22
Antibodies of single specificity (monoclonal) can be obtained by cloning the
appropriate heavy and light chain genes.

35. Figure 6.23d
Engineering to create “immunotoxins”