This video is presented by our volunteer Mehwish Khan, she is from Karachi, Pakistan, and she is covering "Monoclonal Bodies Production via Hybirdoma Technology" for video: https://youtu.be/NilP7HIALvY #Hybirdoma #antibody #monoclonalantibodies #biology #physiology #health #polyclonalantibody

1. TABLE OF CONTENT
1. WHAT IS MONOCLONAL ANTIBODY?
2. WHAT IS ANTIBODY?
3. POLYCOLONAL ANTIBODY VS MONOCLONAL ANTIBODY
4. PRINCIPLE OF MONOCLONAL ANTIBODY
5. HYBRIDOMA TECHNOLOGY
6. PRODEDURE-HYBRIDOMA TECHNOLOGY
7. WHY HAT MEDIUM IS USED?
8. THERAPEUTIC USE OF MONOCLONAL ANTIBODY

2. MONOCLONAL
ANTIBODIES
DEFINITION
ANTIBODIES DERIVED FROM SINGLE CLONE OF PLASMA CELLS; ALL HAVING THE
SAME ANTIGEN SPECIFICITY I.E PRODUCED AGAINST SINGLE EPITOPE OF SAME
ANTIGEN.

3. WHAT IS ANTIBODY?
• ANTIBODIES ARE IMMUNE SYSTEM-RELATED PROTEINS
CALLED IMMUNOGLOBULINS.
• EACH ANTIBODY CONSISTS OF FOUR POLYPEPTIDE
• TWO HEAVY CHAINS AND TWO LIGHT CHAINS JOINED
TO FORM A "Y" SHAPED MOLECULE.

4. MONOCLONAL VS POLYCLONAL
ANTIBODY
POLYCLONAL ANTIBODY
1. Antibodies arise from multiple clone B cell
2. Mixture of antibodies that detect different
epitope on an antigen.
3. Typical in serum in response to antigen.
MONOCLONAL ANTIBODY
1. Antibody from a single parent clone
2. Antibody from one clone detect one
epitope of an antigen
3. Usually produced in a laboratory using
Hybridoma technology.

5. PRINICIPLE OF MONOCLONAL ANTIBODY
PRODUCTION
Clone of B cell stimulated against a single epitope of an antigen is fused with
an Immortal cell e.g. myeloma cell(capable of multiplying indefinitely) to
produce hybridoma cell. This hybridoma cell has two unique properties:
1.Produce monoclonal antibody of same antigen specificity(due to B cell
component.
2.Multiplyies indefinitely producing clone of identical cells(due to immortal cell
component)

6. HYBRIDOMA TECHNOLOGY
PROCEDURE

7. PROCEDURE-HYBRIDOMA TECHNIQUE
MOUSE SPLENIC B CELLS:
Mouse is injected with an antigen containing the desired epitope(s).
After an interval ,the mouse splenic B cells are obtained which would contain the mixture of B
cells activated against the epitope of antigen injected .
MYELOMA CELLS:
Used as source of immortal cells
Myeloma cells are cancerous plasma cells ,closely resemble mouse B cells, hence are
compatible for fusion.
Have capacity to produce their own antibodies.
Hence ,myeloma cells are genetically modified with two mutations(double mutated myeloma
cells)so that they lose the ability to produce their own antibody but retain immortal property.

8. FUSION USING PEG
BROTH
• FUSION-the mouse splenic B
cells and mutated myeloma cells
are fused in polyethylene glycol
broth . In the reaction chamber ,
three types of cells will be
generated
1. Unfused myeloma cells
2. Unfused mouse splenic B cells
3. Fused hybridoma cells
• Purification(by sub culturing on
HAT medium)-the next step is to
remove the unwanted cells and
to propagate the clone of
hybridoma cells . This is carried
out by sub-culturing the cells in
reaction chamber onto special
medium called HAT medium.

9. WHY HAT MEDIUM IS USED?
HAT medium contains:
1. HYPOXANYTHINE
2. AMINOPTERIN
3. THYMIDINE
Purine synthesis in mammalian cell(e.g. splenic B cell)occur by either denovo or salvage
pathways
Aminopterin blocks the de-novo pathway so that cell has to perform the salvage pathway to
synthesize purine for its survival.
Salvage pathway requires two important enzymes-HGPRT(hypoxanthine guanine
phosphoribosyl transferase)and thymidine kinase
So any cell e.g. that lacks HGPRT cannot grown on HAT medium.

10. FATES OF CELLS IN HAT MEDIA
Unfused splenic B cells-can grow but do
not survive long as they are not immortal
Unfused myeloma cells-cannot grow as
they lack HGPRT enzyme to perform the
salvage pathway of purine synthesis
Hybridoma cells-can grow and survive long

11. PROCEDURE-HYBRIDOMA TECHNIQUE
SELECTION OF INDIVIDUAL
HYBRIDOMA CELLS:
If the original antigen used has multiple epitopes
many B cells would fuse with myeloma cells to
produce a mixture of hybridoma cells each
having specificity for one epitope.
Medium containing hybridoma cells is then
diluted into multi-well plates to such an extent
that each well contains only one cells
Hybridoma cells producing the desired
monoclonal antibodies are selected by
radioimmunoassay or ELISA technique using the
specific antigen fragments ,are selectively
proliferated.

12. THERAPEUTIC APPLICATION OF
MONOCLONAL ANTIBODY
THERAPEUTICUSE INCLUDE:
1. Treatment of various inflammatory and allergic diseases and
cancer
The mechanism by which monoclonal antibodies work as
therapeutic
1. Suppress immune system
2. Kill or inhibit malignant cells
3. Inhibit angiogenesis

13. REFERENCES
https://youtu.be/h6sGMaf6Yew
https://med.unr.edu/ddl/technology/monoclonal-antibodies
https://studymind.co.uk/notes/producing-monoclonal-antibodies/
https://youtu.be/Y3GwayzivIE

14. ◦THANK YOU