Synthesis, isolation and importance of monoclonal antibodies

hafiz m wasaeem

1. HAFIZ M WASEEM
UE LAHORE PAKISTAN

2.  AntibodiesareglycoproteinsproducedbyBlymphocytes
(cellsofimmune system)whichneutralizethe
pathogens/antigens
 Blymphocytesarecellsofacquiredimmunesystem
 ImmunesystemisderivedfromLatinterm“IMMUNIS”which
referstoimmunityorprotectionagainstthepathogens
 Itisintegratedsystemoforgans,tissues,cells&cell
products(antibodies,cytokines,etc.)thatdifferentiatesself
fromnon–self&neutralizespotentiallypathogenic
organisms/antigens

3. Innate immune system
•primitive
•non specific
•Various types of cells
Acquired immune
system
•advanced
•specific
•B and T lymphocytes
(give humoral & cell
mediated immunity
respectively)INDUCTION OF AN IMMUNE
RESPONSE
Foreign invaders -viruses, bacteria, allergens, toxins and
parasites-constantly bombard our body.

4. Antibodiesarefoundinextracellular
fluids(bloodplasma,lymph,mucus,
etc.)andonthesurfaceofBcells
Thereareabout10milliondifferent
typesofBlymphocytes
Bcellsonstimulationwillactively
secreteantibodiesandarecalled
plasmacells
Providedefenseagainstbacteria,
bacterialtoxinsandviruseswhich
circulatefreelyinbodyfluids

6. ANTIBODIES
Derived from different B
lymphocytes cell lines
POLYCLONAL. MONOCLONAL.
Derived from a single B cell
clone
Batch to batch variation
affecting Ab reactivity &
titre
Offer reproducible &
inexhaustible supply with
exquisite specificity
Enable the development of
secure immunoassay systems
Not powerful tools for
clinical diagnostic tests

7. Eachantibodyhasatleast
•Twoheavychains
•Twolightchains
VariableRegions:Tworegionsatthe
endofY’sarms.Identicalonthesame
antibody,butvaryinothers
ConstantRegions:Stemofmonomer
andlowerpartsofYarmswhichbindto
complementorcells
Structure of antibody and antigen binding cleft

8. IMMUNOTHERAPY
Treatment of the disease by Inducing, Enhancing or
Suppressing the Immune System
Active Immunotherapy
It stimulates body’s own
immune system to fight
disease
Passive Immunotherapy
It rely on the immune system
components (antibodies)
created outside the body

9. PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Hypoxanthineguaninephosphoribosyl
transferase negative (HGPRT)¯
myelomacellsareselected
HGPRT enzymeenablescellsto
synthesizepurinesbysalvagepathway
OrdinarilyabsenceofHGPRTisnota
problembecauseofdenovopathway
ButcellsexposedtoAminopterinare
unabletousedenovopathwayandare
dependentonsalvagepathwayonly
HGPRT
+
Bcellsarefusedwiththe
myelomacellstoproducehybridoma
andaregrowninHATmedium

10. PRODUCTION OF MONOCLONAL ANTIBODY
Step 1: -Immunization of mice
ANTIGEN (purified
proteins, etc.) +
ADJUVANT
(emulsification)
Serum antibody titre is determined
Titre too low
BOOST
(Pure antigen)
Titre High
BOOST
(Pure antigen)
2 weeks

11. PRODUCTION OF MONOCLONAL ANTIBODY
Step 2: -Removal of spleen for antibody producing B cells
After several
weeks of
immunization
High Ab titre in Serum
Spleen is removed
(source of B cells)

12. PRODUCTION OF MONOCLONAL ANTIBODY
Step 3: -Formation of hybridoma cells
FUSION
PEG
MYELOMA CELLS
SPLEEN CELLS
HYBRIDOMA CELLS
ELISA PLATE
Feeder cells
Growth medium
HAT Medium
1.Platingofcellsin
HAT selective
medium
2.Scanningofviable
hybridomas

13. PRODUCTION OF MONOCLONAL ANTIBODY
Step 4: -Cloning of hybridoma cell lines
A. Clone each +ve Culture
B. Test each supernatant for antibodies
C. Expand +ve clones
Mouse
ascites
method
Tissue
culture
method

14. Cells,celldebris,lipids,andclottedmaterialfromsupernatant
orasciticfluidareremovedbyfiltrationwitha0.45µmfilter
AntibodiesarecommonlyseparatedbyproteinA/Gaffinity
chromatography
mAbsselectivelybindtoProteinA/Gandcanbeafterwards
eluted
Step 5: -Separation & purification of monoclonal antibodies

15. Therapeutic applications
Transplant rejection (Muronomab-CD3)
Cardiovascular disease (Abciximab)
Cancer (Rituximab)
Infectious Diseases (Palivizumab)
Inflammatory disease (Infliximab)
Clinical applications
Purification of drugs, Imaging the target
Future applications
Fight against Bioterrorism

16. Hematologicmalignancies
CD20ishighlyexpressedinB
cellmalignancies(95%in
follicularlymphomas)
LowgradenonHodgkin
lymphomaisbeingsuccessfully
treatedwithrituximab
Rituximabiscombinedwith
chemotherapyforintermediate
gradeordiffuselargecellnon
Hodgkinlymphoma

17. Solidtumors
Solidtumorsdonothavemanyspecific
targets
Edrecolomabtargets17-1Aantigen(Ep-
CAM)incolonandrectalcancerby
ADCC&CDCmethods
Trastuzumabisbeingusedtotarget
HER-2(HumanEpidermalgrowthfactor
Receptor2,CD340)antigeninbreast
cancersaloneorincombinationwith
chemotherapy

18. Muromonab-CD3(OKT3)bindstoCD3moleculeon
surfaceofTcellsandpreventsrejectionoforgans
HumanizedversionsofOKT3showpromiseininhibiting
autoimmunedestructionofbetacellsinType1diabetes
InfliximabbindstoTNF-αandisusedininflammatory
diseasessuchasrheumatoidarthritis
OmalizumabbindstoIgEthuspreventingIgEfrom
bindingtomastcells(allergicasthma)
Abciximabinhibitsclumpingofplateletsbybinding
receptorsontheirsurfaceandhelpfulinpreventing
cloggingofcoronaryarteries