SlideShare a Scribd company logo
1 of 28
Contents: 
Introduction 
History & Development 
Procedure 
Identification & Isolation 
Application 
Persisting Problems 
Improvements 
Summary 
Conclusion
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.
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.
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.
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.
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.
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.
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.
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.
Reclone and cultivate positive clones 
ELISA PLATE
Most common screening 
techniques are ELISA and RIA) 
Low concentration 
(1-20 ug/ml) 
High concentration 
(1-10 mg/ml) 
In- Vitro In- Vivo
• 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.
• 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.
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.
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.
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
 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.
 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.
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.
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
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.
• 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)
Hybridoma Technology & its application in fisheries

More Related Content

What's hot

Vaccine Designing
Vaccine DesigningVaccine Designing
Vaccine DesigningMiccrophage
 
USE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY p
USE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY pUSE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY p
USE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY pEsam Yahya
 
10 safety guidelines for recombinant dna research
10 safety guidelines for recombinant dna research10 safety guidelines for recombinant dna research
10 safety guidelines for recombinant dna researchIndranil Bhattacharjee
 
Aquatic biotechnology
Aquatic biotechnologyAquatic biotechnology
Aquatic biotechnologyShivang Patel
 
Bio-control agents: Insecticidal toxins of Bacillus thuringiensis
Bio-control agents:Insecticidal toxins of Bacillus thuringiensisBio-control agents:Insecticidal toxins of Bacillus thuringiensis
Bio-control agents: Insecticidal toxins of Bacillus thuringiensisManisha G
 
Equipment's used in animal cell culture
Equipment's used in animal cell cultureEquipment's used in animal cell culture
Equipment's used in animal cell cultureSubhalaxmiSwain1
 
Use of microbes in mineral benefication and oil recovery
Use of microbes in mineral benefication and oil recoveryUse of microbes in mineral benefication and oil recovery
Use of microbes in mineral benefication and oil recoveryAprajitaSharma15
 
USE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY A
USE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY AUSE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY A
USE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY AEsam Yahya
 
Cell culture, Different type of cell culture media, types of media
Cell culture, Different type of cell culture media, types of mediaCell culture, Different type of cell culture media, types of media
Cell culture, Different type of cell culture media, types of mediaRajashekar Baldhu
 
Antibody engineering by R.S.Priyengha
Antibody engineering by R.S.PriyenghaAntibody engineering by R.S.Priyengha
Antibody engineering by R.S.PriyenghaPriyengha R.S
 
Monoclonal antibodies
Monoclonal   antibodiesMonoclonal   antibodies
Monoclonal antibodiesPV. Viji
 
ANTIBODY ENGINEEERING ITS APPLICATIONS
ANTIBODY ENGINEEERING  ITS APPLICATIONS ANTIBODY ENGINEEERING  ITS APPLICATIONS
ANTIBODY ENGINEEERING ITS APPLICATIONS sana sana
 
Fermentative production of vitamins and amino acids
Fermentative production of vitamins and amino acidsFermentative production of vitamins and amino acids
Fermentative production of vitamins and amino acidsAshika Raveendran
 
lambda cloning vector
lambda cloning vectorlambda cloning vector
lambda cloning vectorNOMI KhanS
 
Contamination in Cultured Cells
Contamination in Cultured CellsContamination in Cultured Cells
Contamination in Cultured CellsSaniyaMulani1
 

What's hot (20)

Vaccine Designing
Vaccine DesigningVaccine Designing
Vaccine Designing
 
USE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY p
USE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY pUSE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY p
USE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY p
 
10 safety guidelines for recombinant dna research
10 safety guidelines for recombinant dna research10 safety guidelines for recombinant dna research
10 safety guidelines for recombinant dna research
 
Aquatic biotechnology
Aquatic biotechnologyAquatic biotechnology
Aquatic biotechnology
 
Cosmids vector
Cosmids vectorCosmids vector
Cosmids vector
 
Nanobodies
NanobodiesNanobodies
Nanobodies
 
Dot blotting
Dot blottingDot blotting
Dot blotting
 
Bio-control agents: Insecticidal toxins of Bacillus thuringiensis
Bio-control agents:Insecticidal toxins of Bacillus thuringiensisBio-control agents:Insecticidal toxins of Bacillus thuringiensis
Bio-control agents: Insecticidal toxins of Bacillus thuringiensis
 
Synthetic peptide vaccines.pptx
Synthetic peptide vaccines.pptxSynthetic peptide vaccines.pptx
Synthetic peptide vaccines.pptx
 
Equipment's used in animal cell culture
Equipment's used in animal cell cultureEquipment's used in animal cell culture
Equipment's used in animal cell culture
 
Use of microbes in mineral benefication and oil recovery
Use of microbes in mineral benefication and oil recoveryUse of microbes in mineral benefication and oil recovery
Use of microbes in mineral benefication and oil recovery
 
USE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY A
USE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY AUSE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY A
USE OF MICROBES IN MINERAL BENEFICIATION AND OIL RECOVERY A
 
Cell culture, Different type of cell culture media, types of media
Cell culture, Different type of cell culture media, types of mediaCell culture, Different type of cell culture media, types of media
Cell culture, Different type of cell culture media, types of media
 
Antibody engineering by R.S.Priyengha
Antibody engineering by R.S.PriyenghaAntibody engineering by R.S.Priyengha
Antibody engineering by R.S.Priyengha
 
Baculovirus
BaculovirusBaculovirus
Baculovirus
 
Monoclonal antibodies
Monoclonal   antibodiesMonoclonal   antibodies
Monoclonal antibodies
 
ANTIBODY ENGINEEERING ITS APPLICATIONS
ANTIBODY ENGINEEERING  ITS APPLICATIONS ANTIBODY ENGINEEERING  ITS APPLICATIONS
ANTIBODY ENGINEEERING ITS APPLICATIONS
 
Fermentative production of vitamins and amino acids
Fermentative production of vitamins and amino acidsFermentative production of vitamins and amino acids
Fermentative production of vitamins and amino acids
 
lambda cloning vector
lambda cloning vectorlambda cloning vector
lambda cloning vector
 
Contamination in Cultured Cells
Contamination in Cultured CellsContamination in Cultured Cells
Contamination in Cultured Cells
 

Viewers also liked

Cryopreservation
CryopreservationCryopreservation
Cryopreservationmegon94
 
MONOCLONAL ANTIBODIES
MONOCLONAL ANTIBODIESMONOCLONAL ANTIBODIES
MONOCLONAL ANTIBODIESUmair hanif
 
Hybridoma technology
Hybridoma technologyHybridoma technology
Hybridoma technologySijo A
 
Hybridoma technology
Hybridoma technologyHybridoma technology
Hybridoma technologyRitesh ranjan
 
monoclonal antibodies
monoclonal antibodiesmonoclonal antibodies
monoclonal antibodiesNasa Ahmad
 
Monoclonal antibody production
Monoclonal antibody productionMonoclonal antibody production
Monoclonal antibody productionSrilaxmiMenon
 

Viewers also liked (9)

Cryopreservation
CryopreservationCryopreservation
Cryopreservation
 
Monoclonal antibody
Monoclonal antibodyMonoclonal antibody
Monoclonal antibody
 
Animal transformation
Animal transformationAnimal transformation
Animal transformation
 
MONOCLONAL ANTIBODIES
MONOCLONAL ANTIBODIESMONOCLONAL ANTIBODIES
MONOCLONAL ANTIBODIES
 
Hybridoma technology
Hybridoma technologyHybridoma technology
Hybridoma technology
 
Hybridoma technology
Hybridoma technologyHybridoma technology
Hybridoma technology
 
monoclonal antibodies
monoclonal antibodiesmonoclonal antibodies
monoclonal antibodies
 
Genetic engineering in animal cells
Genetic engineering in animal cellsGenetic engineering in animal cells
Genetic engineering in animal cells
 
Monoclonal antibody production
Monoclonal antibody productionMonoclonal antibody production
Monoclonal antibody production
 

Similar to Hybridoma Technology & its application in fisheries

MONOCLONAL ANTIBODYPREPARATION AND EVALUATION
MONOCLONAL ANTIBODYPREPARATION AND EVALUATIONMONOCLONAL ANTIBODYPREPARATION AND EVALUATION
MONOCLONAL ANTIBODYPREPARATION AND EVALUATIONnivedithag131
 
Monoclonal antibody production by hybridoma technology
Monoclonal antibody production by hybridoma technologyMonoclonal antibody production by hybridoma technology
Monoclonal antibody production by hybridoma technologyHasnat Tariq
 
hybridoma technology.pptx
hybridoma technology.pptxhybridoma technology.pptx
hybridoma technology.pptxTejaswiniAsawa
 
Monoclonal antibodies & hybridoma technology
Monoclonal antibodies & hybridoma technologyMonoclonal antibodies & hybridoma technology
Monoclonal antibodies & hybridoma technologyAjay Dominic
 
Hybridoma Technology ( Production , Purification , and Application )
Hybridoma Technology  ( Production , Purification , and Application  ) Hybridoma Technology  ( Production , Purification , and Application  )
Hybridoma Technology ( Production , Purification , and Application ) Sakshi Ghasle
 
hybridomatechnology-biotech pharmacy.pptx
hybridomatechnology-biotech pharmacy.pptxhybridomatechnology-biotech pharmacy.pptx
hybridomatechnology-biotech pharmacy.pptxsurya singh
 
Monoclonal antibodies
Monoclonal antibodiesMonoclonal antibodies
Monoclonal antibodiesMicBio3
 
monoclonal antibodies.pptx
monoclonal antibodies.pptxmonoclonal antibodies.pptx
monoclonal antibodies.pptxSheetalSardhna
 
Monoclonal antibodies explanation .pptx
Monoclonal antibodies explanation  .pptxMonoclonal antibodies explanation  .pptx
Monoclonal antibodies explanation .pptxUVAS
 
Monoclonal antibody (MAb)
Monoclonal antibody (MAb)Monoclonal antibody (MAb)
Monoclonal antibody (MAb)ShritilekhaDash
 
MONOCLONAL ANTIBODY PRODUCTION STRATEGY
MONOCLONAL ANTIBODY PRODUCTION STRATEGYMONOCLONAL ANTIBODY PRODUCTION STRATEGY
MONOCLONAL ANTIBODY PRODUCTION STRATEGYAsh Hassan
 
Hybridomatechnology 160204054435
Hybridomatechnology 160204054435Hybridomatechnology 160204054435
Hybridomatechnology 160204054435ANU RAJ
 
Monoclonal Antibodies
Monoclonal AntibodiesMonoclonal Antibodies
Monoclonal AntibodiesAmeer Ahmed
 

Similar to Hybridoma Technology & its application in fisheries (20)

MONOCLONAL ANTIBODYPREPARATION AND EVALUATION
MONOCLONAL ANTIBODYPREPARATION AND EVALUATIONMONOCLONAL ANTIBODYPREPARATION AND EVALUATION
MONOCLONAL ANTIBODYPREPARATION AND EVALUATION
 
Hybridoma .pptx
Hybridoma .pptxHybridoma .pptx
Hybridoma .pptx
 
MCAB
MCABMCAB
MCAB
 
Monoclonal antibodies
Monoclonal antibodiesMonoclonal antibodies
Monoclonal antibodies
 
Copy of monoclonal antibodies.doc seminar
Copy of monoclonal antibodies.doc seminarCopy of monoclonal antibodies.doc seminar
Copy of monoclonal antibodies.doc seminar
 
Monoclonal antibody production by hybridoma technology
Monoclonal antibody production by hybridoma technologyMonoclonal antibody production by hybridoma technology
Monoclonal antibody production by hybridoma technology
 
Hybridoma cell
Hybridoma cellHybridoma cell
Hybridoma cell
 
hybridoma technology.pptx
hybridoma technology.pptxhybridoma technology.pptx
hybridoma technology.pptx
 
Monoclonal antibodies & hybridoma technology
Monoclonal antibodies & hybridoma technologyMonoclonal antibodies & hybridoma technology
Monoclonal antibodies & hybridoma technology
 
Hybridoma Technology ( Production , Purification , and Application )
Hybridoma Technology  ( Production , Purification , and Application  ) Hybridoma Technology  ( Production , Purification , and Application  )
Hybridoma Technology ( Production , Purification , and Application )
 
hybridomatechnology-biotech pharmacy.pptx
hybridomatechnology-biotech pharmacy.pptxhybridomatechnology-biotech pharmacy.pptx
hybridomatechnology-biotech pharmacy.pptx
 
Monoclonal antibodies
Monoclonal antibodiesMonoclonal antibodies
Monoclonal antibodies
 
monoclonal antibodies.pptx
monoclonal antibodies.pptxmonoclonal antibodies.pptx
monoclonal antibodies.pptx
 
Monoclonal antibodies explanation .pptx
Monoclonal antibodies explanation  .pptxMonoclonal antibodies explanation  .pptx
Monoclonal antibodies explanation .pptx
 
Monoclonal antibody (MAb)
Monoclonal antibody (MAb)Monoclonal antibody (MAb)
Monoclonal antibody (MAb)
 
MONOCLONAL ANTIBODY PRODUCTION STRATEGY
MONOCLONAL ANTIBODY PRODUCTION STRATEGYMONOCLONAL ANTIBODY PRODUCTION STRATEGY
MONOCLONAL ANTIBODY PRODUCTION STRATEGY
 
Monoclonal antibodies
Monoclonal antibodiesMonoclonal antibodies
Monoclonal antibodies
 
Hybridomatechnology 160204054435
Hybridomatechnology 160204054435Hybridomatechnology 160204054435
Hybridomatechnology 160204054435
 
Hybridoma technology
Hybridoma technologyHybridoma technology
Hybridoma technology
 
Monoclonal Antibodies
Monoclonal AntibodiesMonoclonal Antibodies
Monoclonal Antibodies
 

Recently uploaded

Stunning ➥8448380779▻ Call Girls In Panchshil Enclave Delhi NCR
Stunning ➥8448380779▻ Call Girls In Panchshil Enclave Delhi NCRStunning ➥8448380779▻ Call Girls In Panchshil Enclave Delhi NCR
Stunning ➥8448380779▻ Call Girls In Panchshil Enclave Delhi NCRDelhi Call girls
 
Presentation Vikram Lander by Vedansh Gupta.pptx
Presentation Vikram Lander by Vedansh Gupta.pptxPresentation Vikram Lander by Vedansh Gupta.pptx
Presentation Vikram Lander by Vedansh Gupta.pptxgindu3009
 
Recombination DNA Technology (Nucleic Acid Hybridization )
Recombination DNA Technology (Nucleic Acid Hybridization )Recombination DNA Technology (Nucleic Acid Hybridization )
Recombination DNA Technology (Nucleic Acid Hybridization )aarthirajkumar25
 
Natural Polymer Based Nanomaterials
Natural Polymer Based NanomaterialsNatural Polymer Based Nanomaterials
Natural Polymer Based NanomaterialsAArockiyaNisha
 
GFP in rDNA Technology (Biotechnology).pptx
GFP in rDNA Technology (Biotechnology).pptxGFP in rDNA Technology (Biotechnology).pptx
GFP in rDNA Technology (Biotechnology).pptxAleenaTreesaSaji
 
Labelling Requirements and Label Claims for Dietary Supplements and Recommend...
Labelling Requirements and Label Claims for Dietary Supplements and Recommend...Labelling Requirements and Label Claims for Dietary Supplements and Recommend...
Labelling Requirements and Label Claims for Dietary Supplements and Recommend...Lokesh Kothari
 
Biopesticide (2).pptx .This slides helps to know the different types of biop...
Biopesticide (2).pptx  .This slides helps to know the different types of biop...Biopesticide (2).pptx  .This slides helps to know the different types of biop...
Biopesticide (2).pptx .This slides helps to know the different types of biop...RohitNehra6
 
PossibleEoarcheanRecordsoftheGeomagneticFieldPreservedintheIsuaSupracrustalBe...
PossibleEoarcheanRecordsoftheGeomagneticFieldPreservedintheIsuaSupracrustalBe...PossibleEoarcheanRecordsoftheGeomagneticFieldPreservedintheIsuaSupracrustalBe...
PossibleEoarcheanRecordsoftheGeomagneticFieldPreservedintheIsuaSupracrustalBe...Sérgio Sacani
 
STERILITY TESTING OF PHARMACEUTICALS ppt by DR.C.P.PRINCE
STERILITY TESTING OF PHARMACEUTICALS ppt by DR.C.P.PRINCESTERILITY TESTING OF PHARMACEUTICALS ppt by DR.C.P.PRINCE
STERILITY TESTING OF PHARMACEUTICALS ppt by DR.C.P.PRINCEPRINCE C P
 
Call Us ≽ 9953322196 ≼ Call Girls In Mukherjee Nagar(Delhi) |
Call Us ≽ 9953322196 ≼ Call Girls In Mukherjee Nagar(Delhi) |Call Us ≽ 9953322196 ≼ Call Girls In Mukherjee Nagar(Delhi) |
Call Us ≽ 9953322196 ≼ Call Girls In Mukherjee Nagar(Delhi) |aasikanpl
 
Lucknow 💋 Russian Call Girls Lucknow Finest Escorts Service 8923113531 Availa...
Lucknow 💋 Russian Call Girls Lucknow Finest Escorts Service 8923113531 Availa...Lucknow 💋 Russian Call Girls Lucknow Finest Escorts Service 8923113531 Availa...
Lucknow 💋 Russian Call Girls Lucknow Finest Escorts Service 8923113531 Availa...anilsa9823
 
Raman spectroscopy.pptx M Pharm, M Sc, Advanced Spectral Analysis
Raman spectroscopy.pptx M Pharm, M Sc, Advanced Spectral AnalysisRaman spectroscopy.pptx M Pharm, M Sc, Advanced Spectral Analysis
Raman spectroscopy.pptx M Pharm, M Sc, Advanced Spectral AnalysisDiwakar Mishra
 
Biological Classification BioHack (3).pdf
Biological Classification BioHack (3).pdfBiological Classification BioHack (3).pdf
Biological Classification BioHack (3).pdfmuntazimhurra
 
Discovery of an Accretion Streamer and a Slow Wide-angle Outflow around FUOri...
Discovery of an Accretion Streamer and a Slow Wide-angle Outflow around FUOri...Discovery of an Accretion Streamer and a Slow Wide-angle Outflow around FUOri...
Discovery of an Accretion Streamer and a Slow Wide-angle Outflow around FUOri...Sérgio Sacani
 
Analytical Profile of Coleus Forskohlii | Forskolin .pptx
Analytical Profile of Coleus Forskohlii | Forskolin .pptxAnalytical Profile of Coleus Forskohlii | Forskolin .pptx
Analytical Profile of Coleus Forskohlii | Forskolin .pptxSwapnil Therkar
 
Boyles law module in the grade 10 science
Boyles law module in the grade 10 scienceBoyles law module in the grade 10 science
Boyles law module in the grade 10 sciencefloriejanemacaya1
 

Recently uploaded (20)

CELL -Structural and Functional unit of life.pdf
CELL -Structural and Functional unit of life.pdfCELL -Structural and Functional unit of life.pdf
CELL -Structural and Functional unit of life.pdf
 
Stunning ➥8448380779▻ Call Girls In Panchshil Enclave Delhi NCR
Stunning ➥8448380779▻ Call Girls In Panchshil Enclave Delhi NCRStunning ➥8448380779▻ Call Girls In Panchshil Enclave Delhi NCR
Stunning ➥8448380779▻ Call Girls In Panchshil Enclave Delhi NCR
 
Presentation Vikram Lander by Vedansh Gupta.pptx
Presentation Vikram Lander by Vedansh Gupta.pptxPresentation Vikram Lander by Vedansh Gupta.pptx
Presentation Vikram Lander by Vedansh Gupta.pptx
 
Recombination DNA Technology (Nucleic Acid Hybridization )
Recombination DNA Technology (Nucleic Acid Hybridization )Recombination DNA Technology (Nucleic Acid Hybridization )
Recombination DNA Technology (Nucleic Acid Hybridization )
 
Natural Polymer Based Nanomaterials
Natural Polymer Based NanomaterialsNatural Polymer Based Nanomaterials
Natural Polymer Based Nanomaterials
 
GFP in rDNA Technology (Biotechnology).pptx
GFP in rDNA Technology (Biotechnology).pptxGFP in rDNA Technology (Biotechnology).pptx
GFP in rDNA Technology (Biotechnology).pptx
 
Labelling Requirements and Label Claims for Dietary Supplements and Recommend...
Labelling Requirements and Label Claims for Dietary Supplements and Recommend...Labelling Requirements and Label Claims for Dietary Supplements and Recommend...
Labelling Requirements and Label Claims for Dietary Supplements and Recommend...
 
Biopesticide (2).pptx .This slides helps to know the different types of biop...
Biopesticide (2).pptx  .This slides helps to know the different types of biop...Biopesticide (2).pptx  .This slides helps to know the different types of biop...
Biopesticide (2).pptx .This slides helps to know the different types of biop...
 
PossibleEoarcheanRecordsoftheGeomagneticFieldPreservedintheIsuaSupracrustalBe...
PossibleEoarcheanRecordsoftheGeomagneticFieldPreservedintheIsuaSupracrustalBe...PossibleEoarcheanRecordsoftheGeomagneticFieldPreservedintheIsuaSupracrustalBe...
PossibleEoarcheanRecordsoftheGeomagneticFieldPreservedintheIsuaSupracrustalBe...
 
STERILITY TESTING OF PHARMACEUTICALS ppt by DR.C.P.PRINCE
STERILITY TESTING OF PHARMACEUTICALS ppt by DR.C.P.PRINCESTERILITY TESTING OF PHARMACEUTICALS ppt by DR.C.P.PRINCE
STERILITY TESTING OF PHARMACEUTICALS ppt by DR.C.P.PRINCE
 
9953056974 Young Call Girls In Mahavir enclave Indian Quality Escort service
9953056974 Young Call Girls In Mahavir enclave Indian Quality Escort service9953056974 Young Call Girls In Mahavir enclave Indian Quality Escort service
9953056974 Young Call Girls In Mahavir enclave Indian Quality Escort service
 
Engler and Prantl system of classification in plant taxonomy
Engler and Prantl system of classification in plant taxonomyEngler and Prantl system of classification in plant taxonomy
Engler and Prantl system of classification in plant taxonomy
 
Call Us ≽ 9953322196 ≼ Call Girls In Mukherjee Nagar(Delhi) |
Call Us ≽ 9953322196 ≼ Call Girls In Mukherjee Nagar(Delhi) |Call Us ≽ 9953322196 ≼ Call Girls In Mukherjee Nagar(Delhi) |
Call Us ≽ 9953322196 ≼ Call Girls In Mukherjee Nagar(Delhi) |
 
Lucknow 💋 Russian Call Girls Lucknow Finest Escorts Service 8923113531 Availa...
Lucknow 💋 Russian Call Girls Lucknow Finest Escorts Service 8923113531 Availa...Lucknow 💋 Russian Call Girls Lucknow Finest Escorts Service 8923113531 Availa...
Lucknow 💋 Russian Call Girls Lucknow Finest Escorts Service 8923113531 Availa...
 
Raman spectroscopy.pptx M Pharm, M Sc, Advanced Spectral Analysis
Raman spectroscopy.pptx M Pharm, M Sc, Advanced Spectral AnalysisRaman spectroscopy.pptx M Pharm, M Sc, Advanced Spectral Analysis
Raman spectroscopy.pptx M Pharm, M Sc, Advanced Spectral Analysis
 
Biological Classification BioHack (3).pdf
Biological Classification BioHack (3).pdfBiological Classification BioHack (3).pdf
Biological Classification BioHack (3).pdf
 
The Philosophy of Science
The Philosophy of ScienceThe Philosophy of Science
The Philosophy of Science
 
Discovery of an Accretion Streamer and a Slow Wide-angle Outflow around FUOri...
Discovery of an Accretion Streamer and a Slow Wide-angle Outflow around FUOri...Discovery of an Accretion Streamer and a Slow Wide-angle Outflow around FUOri...
Discovery of an Accretion Streamer and a Slow Wide-angle Outflow around FUOri...
 
Analytical Profile of Coleus Forskohlii | Forskolin .pptx
Analytical Profile of Coleus Forskohlii | Forskolin .pptxAnalytical Profile of Coleus Forskohlii | Forskolin .pptx
Analytical Profile of Coleus Forskohlii | Forskolin .pptx
 
Boyles law module in the grade 10 science
Boyles law module in the grade 10 scienceBoyles law module in the grade 10 science
Boyles law module in the grade 10 science
 

Hybridoma Technology & its application in fisheries

  • 1.
  • 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.
  • 5.
  • 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.
  • 12.
  • 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.
  • 17.
  • 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.
  • 24.
  • 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)