New arena for fish biotechnology

2. Contents:
Introduction
History & Development
Procedure
Identification & Isolation
Application
Persisting Problems
Improvements
Summary
Conclusion

3. Introduction
• Hybridoma technology is one kind of
Biotechnology.
• It is a hybridization technique which is used to
produce antibody producing hybrid cell.
• These hybrid cells are produced by fusing the
genetic material of B-lymphocyte with tumour cell.
• The antibodies produced are monoclonal
antibodies as they are all of a single specificity.
• Presence of B-lymphocyte genetic material helps
in antibody production, whereas capacity to divide
indefinitely in the culture due to the presence of
tumour cell.

4. The production of monoclonal antibodies was invented by Cesar
Milstein and Georges J. F. Köhler in 1975.
They pre-programmed B-lymphocytes to respond to a single type of
antigen or antigenic determinant, therefore they produce single type of
antibody specific to the specific antigen.
When an antigen reacts with B-lymphocyte receptors, lymphocytes
divide rapidly and produce a clone of B cells, all these B cells produce
antibodies against that specific antigen and this is called as clonal
selection. That is B-lymphocytes produce only one type of antibodies
which are specific to only one type of antigen or antigenic determinant.
But fully differentiated antibody producing B-lymphocyte cells known
as plasma cells does not divide when cultured in a laboratory.
The term hybridoma was coined by Leonard Herzenberg during his
sabbatical in Cesar Milstein's laboratory in 1976/1977.

6. Step-I
Mouse is immunized by giving
antigen injection against which
monoclonal antibodies have to
produced along with an
adjuvant. This is followed by
booster doses of the antigen.
After 72 hrs of immunization the
spleen is collected from the
mouse.
Then a free cell suspension
medium of the spleen is
prepared using sterile serum
free medium followed by
centrifugation.
All these processes are applied
for extraction of plasma cells
from the spleen.

7. Step-II
Maintain the plasma cells in
the cell culture medium for
16-24 hrs before fusion.
It should be ensured that the
cells are in early phase of
growth at the time of fusion.
The myeloma cells are
selected based on some criteria
like these cells themselves
should not produce
antibodies and also they
should contain a genetic
markers such as
HGPRT(hypoxanthine-guanine
phosphoribosyltransferase) .
This genetic marker helps in
easy selection of the resulting
hybrid cells.

8. Step-III
At the time of fusion both
myeloma & spleen cells are
counted & then mix in the
appropriate ratio.
Depending on the properties of the
tumour cell, the mixing ratio of
spleen to tumour cell may vary
from 5:1 to 2:1
Following the mixing, the cells are
centrifuged into a loose pellet.
The supernatant is removed & the
pellet is mixed with 1 ml of
Polyethylene glycol(PEG) for 3
min.
In doing so the pellet will be broken
up into uniform small clumps.

9. Step-IV
• Following the fusion, dilute
the cells in serum free
medium slowly to reduce
the osmotic disruption of
the fused cell.
• Then centrifuge the cells &
resuspend in HAT
(Hypoxanthine Aminopetrin
Thymidine) medium.
• Then Hat medium containing
the fused cells are allowed
to incubate in a CO2
incubator for 3-4 days.

10. Step-V
• This mixture of cell population
is then cultured in selective
media known as HAT medium
along with the drug
aminopterin.
• The HGPRT myeloma cells
cannot divide in the HAT
medium due to the presence
of aminopterin.
• The Specific antibody
producing B-lymphocytes are
unable to divide continuously
in the culture medium,
therefore eventually they die.

11. Step-V
• Only the hybridoma cells
have got the ability to
divide and proliferate on
the HAT medium because
genome from the B-lymphocyte
makes them
HGPRT positive and
genome from the myeloma
cells they can divide
indefinitely.
• Thus only the hybridoma
cells or fused cells are
selected using selective
media called as HAT
medium.

13. Reclone and cultivate positive clones
ELISA PLATE

14. Most common screening
techniques are ELISA and RIA)
Low concentration
(1-20 ug/ml)
High concentration
(1-10 mg/ml)
In- Vitro In- Vivo

15. • The mouse ascites method usually produces
very high mAb concentrations that often do not
require further concentration procedures that can
denature antibody and decrease effectiveness.
• The high concentration of the desired mAb in
mouse ascites fluid avoids the effects of
contaminants in in -vitro batch-culture fluid when
comparable quantities of mAb are used.
• The mouse ascites method avoids the need to
teach the antibody producer tissue-culture
methods.

16. • The mouse ascites method involves the
continued use of mice requiring daily
observation.
• MAb produced by in vivo methods can contain
various mouse proteins and other
contaminants that might require purification.
• The mouse ascites method can be expensive if
immuno-deficient mice in a barrier facility
used.
• In vivo methods can cause significant pain or
distress in mice.

18. In vitro methods reduce the use of mice at the antibody-production
stage but can use mice as a source of feeder cells
when antibody generation is under way).
In vitro methods are usually the methods of choice for large-scale
production by the pharmaceutical industry because of
the ease of culture for production, compared with use of
animals, and because of economic considerations.
In vitro methods avoid the need to submit animal protocols
to IACUCs.
In vitro methods avoid or decrease the need for laboratory
personnel experienced in animal handling.
In vitro methods using semipermeable-membrane-based
systems produce mAb in concentrations often as high as those
found in ascitic fluid and are free of mouse ascitic fluid
contaminants.

19. It should be noted that each of the items below pertains to only a
fraction (3–5%) of hybridomas, but they indicate some of the
difficulties associated with in vitro methods.
Some hybridomas do not grow well in culture or are lost in
culture.
The loss of proper glycosylation of the antibody (in contrast
with in vivo production) might make the antibody product
unsuitable for in vivo experiments because of increased
immunogenicity, reduced binding affinity, changes in biologic
functions, or accelerated clearance in vivo.
In general, batch-culture supernatants contain less mAb
(typically 0.002-0.01) per milliliter of medium than the mouse
ascites method. Note that semipermeable-membrane-based systems
have been developed that can produce concentrations of mAb
comparable with concentrations observed in mouse ascites fluid.

20. Dose determination of a medicine
To detect allergies & viral disease
 to detect certain type of cancer; to monitor the
presence or appearance of malignant cells after
surgical or radio-therapeutic treatments.
 For purification of complex biological mixture.
Envisaged for the labelling & precise
identification of some specialized cell such as
neurone for gaining better knowledge of the way
of association & operation.
 For purification of structural cell membrane
protein.
Mab has a great role in serotherapy

21.  Used to produce Immunotoxin
 Used in the preparation of very specific vaccines,
particularly against certain virus strain or against
some parasites.
 Neutrilize the action of lymphocytes responsible for
the rejection of grafts & destroy the auto-antibodies
produced in auto-immune disease.
 Mab could considerably increase the effectiveness
of the medicinal substances on the target cells,
while avoiding the serious side-effects of cancer
therapies.

22.  The production of monoclonal antibodies remains
inefficient and labor intensive
 The process of immortalizing a B cell is so inefficient that
only 1 in 20,000, or sometimes 1 in 200,000, B cells forms
a viable hybridoma.
 Generation of useful monoclonal an- tibodies to weak
immunogens are very difficult as If the antigen is poorly
immunogenic, available in only small amounts, or impure,
the numbers of B cells making antibody to that antigen will
be low and only a few positive hybridomas will be
identified.
 The efficiency of fusion decreases greatly if one waits until
the end of the immune response. when B cells are no
longer proliferating rapidly and synchronously, even though
this is when the highest affinity antibodies are being
made.

23. IMPROVEMENTS IN THE
HYBRIDOMA TECHNOLOGY
 Enrichment of the cells making the antibody(B- Cells) of
interest using in vitro immunization would reduce the amount
of screening and make it possible to start with larger
populations of cells and immortalize the more number of B
cells.
 In one version of this technique, immune cells are incubated
with antigen-biotin conjugates and then mixed with biotin-coated
myeloma cells. Avidin is added to bring the B cells and
the fusion partner(Tumour Cell) together and they are then
fused by electrofusion
 When antibody-forming cells are grown in culture, most of the
B cells die, but those that are stimulated by antigen will
proliferate and over- grow than the rest of the population.

25. 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

26. Conclusion
Many European & North-American firms are
interested in the applications of Mab. In california,
for-instance, certain medicine companies are
preparing diagnostic kits designed for the
screening of certain lethal diseases. It is
anticipated that the future support of some
aspects of this hybridomas based monoclonal
antibody technology will be tailored to the needs
of aquaculture industry of each developing
country of the world.

27. • http://www.medarex.com/Development/UltiMAb.htm
• http://wwwext.amgen.com/media/media_pr_detail.jsp?y
ear=2006&releaseID=837754
• http://www.regeneron.com/velocimmune.html
• http://www.ukbusinesspark.co.uk/cay92125.ht
• http://www.biotecharticles.com/Others-
Article/Hybridoma-Technology-A-Biotechnology-
Technique-378.html
Links:
Book:
Fish Biotechnology by Ranga &
Shammi (Pg No. 230-237)