Hybridoma technology/ Monoclonal antibodies

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1. Hybridoma Technology
Presented by
Miss Pratiksha G. Puranik
M.Sc. (Zoology) B.Ed. Ph.Dpursuing
Shri Shivaji Science College Amravati

2. Contents
Introduction
Immunizationofanimals
Isolationofstimulatedspleencells
Myelomacellslinesusedasapartner
Fusionmethods
Detectionofmonoclonalantibodies
Applicationsofmonoclonalantibodies
References

3. Introduction
In1975,GeorgesKöhlerandCesar
Milsteindevisedamethodforpreparing
monoclonalantibodies.
Itisainvitrotechnologyofforminghybrid
celllinescalledashybridomas.
Ahybridomaisahybridcellobtainedby
fusionofB-cellusuallywithatumourcells
(myelomas).
Thesehybridomaspossessesabilityto
produceantibodyduetoB-cellgenome&
capacitytogrowindefinitelydueto
myelomacells.
Therefore,specifichybridomasarecultured
invitroormaypassedthroughperitoneal
cavityofmousetoobtainmonoclonal
antibodies.
Monoclonalantibodiesarederivedfroma
singleclone&thereforearespecificfor
epitopeofantigen.
Milstein and Kohler

4. Immunization of animals
Miceareimmunizedwithrepeatedinjectionofantigenwhich
maybewholecell,membranefragmentorcomplexmolecules.
Thismicethenproducesantibodyagainstthatinjectedantigen
whichistestedbyELISA.
Spleenisthenremovedasasourceofcellforcellfusion.

5. Isolation of stimulated spleen
cells
Thespleenfromimmunizedmicei.e.,stimulatedspleenis
removed&spleenocytesareculturedseparately.
Thisculturedspleenocytesthenproducesantibodies&are
fusedwithmyelomacells.

6. Myeloma cells used as a fusion
partner
Generally,myelomacellsareusedasafusionpartnerbecause
thespleenocytesoranynormalcellhasspecificlife-span&
theydiesinculturemediumwhentheirlife-spanisfinished.
Toproducemonoclonalantibodies,continuallydividingcells
arerequired.
TheylacksHGPRTgene.

7. Fusion methods
PEGorSendaivirusorelectricalshocksaresomeofthefusion
agents,causingfusionofspleenocyteswithmyelomacells
whichmakestheircellmembranesmorepermeable.
FusedcellsareincubatedinHATmediumfor10-14days.
Thisfusionresultsintheformationofhybridoma.

10. Detection of monoclonal
antibodies
Desiredhybridomasareselectedforscreening.
Thismediumisdilutedintomulti-wellplatessuchthateach
wellwillcontainonlyonecell.
Sinceantibodiesinawellareproducedbysamespleencell(B-
cellofspleen)theywillbemigratetowardstheepitopeof
injectedantigen&thusamonoclonalantibody.

11. Applications
Monoclonal antibodies are used for –
Detecting pregnancy, diagnosing numerous pathogenic microorganisms.
Measuring the effect of various drugson bloodlevels .
Matching histocompatibility antigens, and detecting antigens shed by certain
tumors.
Radiolabeled monoclonal antibodies can also be used in vivo for detecting or
locating tumor antigens, permitting earlier diagnosis of some primary or
metastatic tumors in patients.
For classification of blood groups.

12. Immunodiagnosis:-Thediagnosisofmanyinfectious
diseasesreliesonthedetectionofparticular
antigensorantibodiesinthecirculationorintissuesbyuse
ofmonoclonalantibodiesinimmunoassays.
Functionalanalysisofcellsurfaceandsecretedmolecules.
Monoclonalantibodiesarealsowidelyusedtopurifyselectedcellpopulations
fromcomplexmixturestofacilitatethestudyofthepropertiesandfunctions
ofthecells.

13. References
1.Kindt,T.J,KubyJ.GoldsbyA.,OsborneB.A,ImmunologyVEdition.
Pp:99-101
2.Abbas,A.K,LichtmanA.H.,PillaiS.CellularandMolecularImmunology
VIIEdition.Pp97-98