4. OBjECTIvES
• Hybridoma technology
• To understand concept behind production of monoclonal
Antibodies
• To understand various steps in production of hybridomas
• To understand method for preparation of hybridomas
• Role of hybridmas in plants
• Application of hybrdioma technology
8. HISTORY
o Developed by Georges
J.F. Kohler and Cesar
Milstein in 1975
o They shared nobel prize
for this discovery in 1984
o The term hybridoma was
coined by Leonard
Herzenberg in 1975
9. PRINCIPLE
o The hybrid cell has the capacity of antibody
production derived from B cells
o At the same time it can divide continuously
by the quality derived from Myeloma cells
o By combining the desired qualities of both
the cells, the technology ensures large scale
Antibody production of single specificity
10. WHAT ARE ANTIBODIES?
An antibody is a
protein used by
the immune
system to
identify and
neutralize
foreign objects
like bacteria and
viruses.
11. MONOCLONAL ANTIBODIES
• Monoclonal antibodies (mAb) are
antibodies that are identical because
they are produced by one type
of immune cell, all clones of a single
parent cell.
14. PRODUCTION Of MONOCLONAL
ANTIBODIES
• Mixture of fused and unfused cells
• Myeloma cells lack HGPRT or TK thus
produced in HAT medium Plan cells
• Colonial expansion
18. REqUIREMEnT of HybRIDoMA
lAb
• CO2 incubator
• Inverted microscope
• -78 `c deep freeze
• Centrifuge
• Liquid nitrogen container
• 25 cm2 or 75 cm2 tissues cultures flask
• Penicillin and streptomycin
19. TIssUE cUlTURE flAsk
• Rectangular and have tilted
neck
• Designed to provide
maximum surface for
growth of cells.
• polypropylene or
polystyrene
• it have three size via
25cm, 75cm2 and 225 cm2
20. 96 WElls TIssUE cUlTURE
plATEs
• 96 well plates and well flask
with bottom volume 400-500
micro liters
• Transparent and symmetrical
wells plates.
• wells plates are at defined
positions.
• 8 rows and 12 columns
• well designed
• number e.g. A3,B5,C6 ,D4 etc
21. MyEloMA cEll cUlTURE
MEDIUM
• Cultured in RPMI-1640 or DMEM
• Basal medium containing 10 to 15% FCS.
• Basal medium contains salts, glucose, vitamins,
phenol red and calf serum.
• Penicillin and streptomycin.
22. pREpARATIon of RpMI-1640
• 1 liter of medium is prepared by dissolving
following intergradient's in distilled water.
• RPMI-1640 1 vial
• NaHCO3 2-20 g
• HEPES 2-38 g
• Sod pyruvate 0.11 g
• Glutamine 0.57 g
• penicillin 100 U/ml
• streptomycin 0.10 g
23. MyEloMA cEll cUlTURE
• Revived from frozen stock or burrowed or purchased.
• Cells on arrival are fit with fresh basal medium.
• 15 FCS & 5% co2 and 37'c
• Phenol red indicates the pH of the medium.
• If medium turns yellow and is transparent it indicates that
medium is to be replenished.
• If medium turns yellow and is turbid it indicates that bacterial
or fungal growth has occurred.
24. conT….
• Splitting cells 1:9 times with fresh medium.
• Confluence term is for extent surface
occupied by growing cell mass
• It is appropriate to sub-culture cells at
70-90 % confluence.
25. HARvEsTIng of MyEloMA
cElls
• Myeloma cells growing in log phase of 3rd
generation are used for fusion.
• Cells are centrifuged at 1000 ppm for
5minuts.
• Cells are counted after staining with trypan
blue dye.
26.
27. cElls cUlTURE
conTAMInATIon
• Medium also support growth of bacteria
fungal and mycoplasma
• They divide at much faster rate then myeloma
cells
• Deplete nutrients very fast.
• Contaminations come from environment,
during culture handling and prepration of
28. sTEpWIsE pRocEDURE
• o Isolation of B cells
• -Mice , 2-4 weeks old are immunized with the
antigen against which monoclonal antibodies
are to be raised by subcutaneous injection
• -Later B cells are isolated from the spleen of
an immunized mouse
29. soMATIc cEll fUsIon
• -Electrofusion : cells are allowed to fuse
with the application of an electric field
• -Done by using PEG medium
• -PEG stands for Poly Ethylene Glycol
30.
31. Selection of hybrid cellS
•-HAT medium is used for the selection
of hybrid cells
•-HAT stands for Hypoxanthine
Aminopterine Thymidine
32. how hAt medium workS in the
Selection of hybrid cellS
• -B cells are HGPRT+ and can survive in the HAT
medium, but they undergo normal cell death after
some division
• - In hybridoma technology, the myeloma cells used
are HGPRT deficient
• -So these cells can’t survive in HAT medium as
Aminopterine blocks the Denovo pathway
33. cont..
o Hybrid cells has HGPRT enzyme from the B
cell as well as they have the ability to
multiply repeatedly as myeloma cells
o So only hybrid cells can survive in HAT
medium
34.
35. identificAtion And iSolAtion of
the hybridomA cellS
• The first screening technique used is ELISA
• -Done by incubating the hybridoma culture
supernatant, secondary enzyme labeled
conjugate and chromogenic substrate
• -Formation of a coloured product indicates a
positive hybridoma
36. cont…
• Two methods have been used for multiplying
the hybridoma cells
1.In-vivo
2.In-vitro
37.
38. cont…
o Iv-vivo procedure involves introduction of
hybridoma cells into the peritoneal cavity of
the animal ,then ascetic fluid is isolated and
then antibodies are isolated from it
o In-vitro method involves culturing of
hybridoma cells in suitable culture media and
then antibodies are isolated and purified
39. cont..
• Once a hybridoma colony is established, it
will continually grow in culture medium like
RPMI-1640 and produce antibodies
40. multiwell plAteS
• Multiwell plates are used initially to grow the
hybridomas
• After selection, they are changed to tissue culture
flasks
• This provides enough cells for cryopreservation and
supernatant for subsequent investigations
• The supernatant can yield 1 to 60micrograms per ml
which can be maintained at lower temperatures for
future use
42. monoclonAl AntibodieS in
plAntS
• A plantibody is antibody/proteins
produced by genetically modified crops.
• Uses: as edible vaccines, diagnostic/
therapeutic monoclonal antibodies, for
disease resistance in plants.
45. PLANT BREEDING BY
SEXUAL CROSS
• Advantages:
New antibody combinations
can be produced.
Dual purpose vaccines can be made.
Properties like enhanced expression, stability, binding
affinity can be achieved using different promoters,
signal sequences.
Mutations in CDRs of V- regions can enhance binding
affinity of antibodies.
46. PLANT TISSUE CULTURE
ADVANTAGES:
• Cell cultures contain fewer biological proteins or molecules
which may contaminate the product.
• Large amounts of proteins obtained in short time & less
purification steps.
• Sexual reproduction is not needed to ensure the lifespan of the
species.
• Transgene stability is also increased.
47. APPLICATIONS
Treatment of infectious disease,
inflammation, autoimmune disease or cancer.
Tobacco produced mAb is more viable
alternative to mAb produced in mouse ascites
fluid for the large amounts needed for
purification of hepatitis B vaccine.
48. CONT..
World's first clinically tested plantibody, CaroRx
binds specifically to Streptococcus mutans, the
bacteria that cause tooth decay, and prevents the
bacteria from adhering to teeth.
CaroRx is intended for regular topical preventative
administration by both dental hygienists and patients
allowing a thorough cleaning and intervention for any
existing decay.
50. FuNCTIONAL
ANTIbOdIeS Need
• Need to be properly folded and assembled.
• Need disulfide bond formation and
glycosylation.
• Glycosylation is different in plants.
• Only mannose is attached-shorter half-life of
Ab.
51. CONCLuSION
Hybridomas are cells that have been engineered to produce a
desired antibody in large amounts, to produce monoclonal
antibodies.
Monoclonal antibodies can be produced in specialized cells
through a technique now popularly known as hybridoma
technology
The advantage of this process is that it can combine the
qualities of the two different types of cells; the ability to grow
continually, and to produce large amounts of pure antibody.
52. Cont..
• Production of monoclonal antibodies
• Selective production of stereospecific monoclonal
antibodies
• Efficient generation of human monoclonal antibodies
for clinical purposes.
53. CONT..
• Hybridoma technology have changed the
paradigm of plant as a food source to so-
called plant bioreactor to produce valuable
recombinant proteins. These include
therapeutic or diagnostic monoclonal
antibodies, vaccines, and other
biopharmaceutical proteins.
54. ReFeReNCeS
• Monoclonal antibody engineering in
plants
• •Andrew Hiatt , Julian K-C. Ma
• •Department of Cell Biology, The Scripps Rcsearch Institute, La
Jolla, CA 92037, USA
• Plantibodies: applications, advantages and bottlenecks
• Molecular Biotechnology Unit, John Innes Centre, Norwich
Research Park, Norwich
• NR4 7UH, UK
55. CONT..
• Milstein, C (1999). "The hybridoma revolution: an offshoot of
basic research“
• Nelson, PN; Reynolds, GM; Waldron, EE; Ward, E; Giannopoulos,
K; Murray, PG (2000). "Demystified …: Monoclonal
antibodies". Molecular pathology
• Ghosh, AK; Mason, D Y; Spriggs, A I (1983).
"Immunocytochemical staining with monoclonal antibodies in
cytologically.
• https://www.sciencedirect.com/topics/immunology-and.../hybridoma-
technology
• https://www.ncbi.nlm.nih.gov
56. Department for Molecular Biotechnology, RWTH Aachen,
Worringerweg 1, 52074 Aachen, Germany
PLANTIBODY: AN OVERVIEW(Asian journal of Pharmacy
and Life Science, Vol. 1 (1), Jan-Mar, 2011)
Priya Jain*, Prasoon Pandey, Dheeraj Jain, Pankaj Dwivedi
College of pharmacy, IPS academy, rajendra nagar, Indore,
India. 452012
