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1. Monoclonalantibody
By
Miss. Supriya. S. Daphal
F.Y .M . Pharm
Under the Guidance of
Mr. D. M. Ghadge
(M. Pharm pharmaceutics )
Gourishankar Institute of Pharmaceutical Education &
Research Limb, Satara.
(2017-18)
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2. CONTENT
 Introduction
 Methods of preparation
 Advantages
 Disadvantages
 Application
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3. INTRODUCTION
 Antibodies are part of defense system that can protect body against the invading
foreign substance namely antigen
 Antibodies are glycoprotein molecule present in the serum
 They are produced in response to antigen that are protein or polysaccharides
molecule and may be foreign substance to the body .
 Antibodies are selected class of blood cells known as B- lymphocytes which form
clone of cell known as plasma cell
 Antibodies classified into five main types –such as IgG,IgM, IgA , IgE,IgD
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4. INTRODUCTION
 In response to antigen B –lymphocytes gear up and produce different antibodies.
1] Monoclonal antibody (mAbs)
Single type of antibody that is directed against a specific antigenic determinant .
2] Polyclonal antibody ( PoAbs )
These type of antibodies will react with same antigen .
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5. MONOCLONAL ANTIBODY
 G.Kolher and C.Milstein (1975)first reported production of mAbs.
 First production of mAbs is an amalgamation of immunochemistry in –vitro
cultivation of cancer cell .
 A new era immunology was launched in 1975 with discovery of hybridoma
technique (method of creating pure antibodies against a specific antigen )
 Kolher and Milstein (1975) succeed in fusing a normal B- cell with a
myeloma cell known as hybridomas .
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6. METHOD OF PREPATRATION
Method of preparation of monoclonal antibody involves six stages.
1. Immunization
2. Cell fusion
3. Formation and selection of hybrid cell
4. Screening of product
5. Cloning and propogation
6. Characterization and storage
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8. IMMINIZATION
Immunize animal with appropriate antigen
Antigen along with adjuent or complete adjuvent injected
subcutaneously
Serum concentration was optimum then animal was killed .
Slpeen is aseptically removed
Disrupted by mechanical or enzymatic method to release the cell .
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9. CELL FUSION
Splenocytes mixed with plasmocytoma cell in an appropriate
medium.
This mix is exposed to high concentration of PEG for short time
fusion is alow to take place over a period of time .
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10. Why mouse is used in production of mAbs ?
 Mouse is an inexpensive animal .
 Several billion cell can be obtained from mice spleen.
 The use of HGPRT ( hypoxanthine guanine phosphoribosyl
transferase)cells assure that only one hybridoma cell is selected.
 After 10 days culture in the hypoxanthine aminoprotein
thymidine (HAT)medium , mostly containing dead cells but
few wells containig viable cells .
 Well containing viable cluster are screened for production of
mAbs .
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11. FORMATION AND SELECTION OF HYBRIDOMA
CELL
When cells are cultured in HAT medium ,only hybridoma cell grows
and reste are slowely disappear .
After fusion hybrid cells must be separated from parent cell .
When PEG or sandai virus used as a fusing agent small %of cell is
fused and some of fused cells are homogeneous i.e A-A, B-B cells
These selection depend upon the synthesis of nucleotides and
mammalian cells.
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12. SCREENING OF PRODUCT
Hybridoma cell producing desired antibody secreted by
hybrid cell are known as mAbs
Techniques uesd are
1]ELISA ( enzyme linked immunosorbent assay )
2] RIA ( radioimmunoassay )
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13. CLONING AND PROPOGATION
1] DILUTION METHOD
suspension of hybridoma culture is prepared
Diluted into new wells
only one cell found in well
Cells are grown and procedure are repeated several time to
increase the probability that all the cell in given well are
monoclonal .
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14. 2] SOFT AGAR METHOD
Hybridoma cells cultured on soft agar
fact
Many malignant cell shall proliferate forming colonies in a
semisolid medium containing low quality agar .
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15. CHRACTERISATION AND STORAGE
 Subjected to biophysical and biochemical characterization
 Stability of the cell lines important feature .
 Cells are required to characterize for their ability to freezing
and thawing .
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16. ADVANTAGES
1. In –vitro In-vivo production is possible .
2. High reproducibility .
3. One antigenic determinant .
4. High avidity can produced .
5. Production of cell lines to individual component of mixture is
possible .
6. Determinant to very high value of antiserum titre .
7. Radiolabelling and conjugation fluorescent is possible .
8. The immunization and labelling maintanance of foam or
animal are not required
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17. DISADVANTAGES
1. Method is time consuming and initial cost involved is more.
2. The energy of binding to antigen is slow in mAbs.
3. Not a standard double immunodiffusion method .
4 For ethical reason human cannot be immunized against
antigen.
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18. APPLICATIONS
A] Diagnostic applications.
B] Investigational and analytical applications .
C] Miscellaneous applications .
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19. A] DIAGNOSTIC APPLICATIONS
 mAbs important diagnostic reagent used in biomedical
research , microbiology research in diagnosis of hepatitis ,
influenza , AIDS ,herper simplex and in treatment of cancer .
 More than 100 diagnostic mAbs avialable in market .
 mAbs kit are useful in identification of communicable disease
including transfusion , transmissible infection.
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20. 1. In cardiovascular disease :
it is possible to detect the location and the degree of damage
to heart by using radiolabbled antimyosin mAbs
2. Site of bacterial infection :
this is possible by directing mAbs against bacterial antigen .
3. In cancer :
mAbs having important clinical application in detection and
early diagnosis of cancer , in staging procedure and therapeutic
trial . Capacity to distinguish different type of human tumour .
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21. 4. In treatment of AIDS :
immunosupprescent is the hallmark of AIDS .
This is caused by reduction in CD4 cell of T lymphocytes .
The HIV binds to specific receptor on CD4 cellsby using surface
membrane glycoprotein .
5. In maleria :
immunological process for determination of plasmodium such as
ELISA is rapid .
6. In diadetes :
hybridoma produced mAbs against insulin for insulin assay because
of its specificity and plentiful supply . The resulting anti-insulin
antibody is purified and characterized by RIA
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22. 7. Rheumatic arthritis :
 arthritis is an autoimmune assay .
 mAbs are directed against T lymphocytes and B lymphocytes .
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23. B] Investigational and analytical application
1] Identification and isolation of lymphocytes phenotyping :
This characteristics can be utilized for identification of various stages
of lymphocytes differenciation
Ex : human and mice in both CD8 membrane expressed by T helper cell
2] Purification of protein :
purified antibody added to crude mixture of protein
Specific protein combines with antibody
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24. C] Miscellaneous applications
1] Application for catalytic antibody :
 Wide application in biology, medicine chemistry , and biotechnology .
 mAbs with enzyme linked catalytic properties have been sucessfully raised .
 The catalyse shows various reaction – hydrolysis of ester, amide , and
carbonate , cyclization reaction , biomolecular reaction , elimination reaction
asymmetric synthesis .
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25. 2] mAbs in drug targeting :
 mAbs gainig improtance because of their high specificity .
 In cancer therapy , cytotoxic agents used to kill malignant cell
also damage normal cell.
 mAbs generated against specific antigen when conjugation to
cyto- toxic drugs can selectively deliver to cancer cell while
minimizing damage to normal cells.
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26. Generic name Brand name Manufacturer
Infliximab Remicade Johnson and
Johnson
Transzumab Herceptin Abbott
Cefuximab Erbutux Bristol , Myers and
Merk
Ranbizumab Lucentis Novartis Roche
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27. REFERENCES
 Textbook of Microbiology by Kokate and Jalapure.
 Production of monoclonal antibody by Nandkishor Jha.
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28. THANK YOU
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