Various diagnostic tools now a days relied on the Hybriodoma technology and monoclonal antibodies,so this presentation will give some basic information about mAb and Hybridoma technology.

1. Asst.prof .Dnyaneshwari Joshi.
Department of Biotechnology
and winetechnology

2. • First immortal monoclonal Ab’s produced by George
Kohler and Cesar Milstein in 1975
• They shared Nobel prize for this discovery in 1984
• The term Hybridoma was coined by Leonard
Herzenberg in 1975.

3. • ANTIBODY:
It is a soluble globular protein ( Glycoprotein),Which is produced
in response to a specific antigen and binds via non-specific interactions .
The term Immunoglobulin is used to describe any Antibody
regardless of its specificity , whereas Antibody describes ANTIGEN-
SPECIFIC immunoglobulin.
• MONOCLONAL ANTIBODY(MAb) :
Single type of Antibody which is directed against specific type
of antigenic determinant (epitope) is called as monoclonal antibody.

4. MONOCLONAL ANTIBODIES….
• Antibodies produced from a single clone of B cells.
• Produced by fusing a B cell secreting the desired antibody with
a myeloma cell capable of growing indefinitely in tissue culture.
• Monoclonal antibodies all have identical antigen-binding sites.
Thus they all bind to the same epitope with the same affinity.
They are all of the same antibody class (isotype).

6. 1. Murine antibody.
1.Whole of the antibody is of murine origin
Major problems associated with murine
antibodies include
reduced stimulation of cytotoxicity
Formation of complexes after
repeated administration
allergic reactions.
2. Chimeric antibody.
1.Chimeric antibodies are composed of
murine variable regions fused onto human
constant regions.
2.Antibodies are approximately 65%
human.
3.Humanized antibody.
1.Humanized antibodies are produced by
grafting murine hyper variable amino acid
domains into human antibodies.
2.This results in a molecule of approximately
95% human origin

8. HYBRIDOMA TECHNOLOGY
A hybridoma is a hybrid cell obtained by fusion of B lymphocyte with usually a tumor cell of
antibody forming system or B lymphocyte (these are called myelomas)

9. The hybrid cell has the capacity of antibody production derived from B cells
At the same time it can divide continuously by the quality derived from Myeloma cells
By combining the desired qualities of both the cells, the technology ensures large scale
Antibody production of single specificity
Specific hybridomas are either cultured in vitro or passed through mouse peritoneal cavity
to obtain monoclonal antibodies, this is called as hybridoma technology
PRINCIPLE

10. 1.Isolation of B cells
-Mice , 2-4 weeks old are immunized with the antigen against which monoclonal antibodies are
to be raised by subcutaneous injection
-Later B cells are isolated from the spleen of an immunized mouse
2.Isolation of myeloma cells
-Myeloma cells are isolated from bone marrow
-The myeloma cells used are HGPRT(Hypoxanthine-guanine phosphoribosyl transferase) mutant
cells ( raised by mutations using 8-azaguanine)
STEPWISE PROCEDURE

11. • This Cell Line Is Deficient In HGPRT (hypoxantine guanine
phosphoribosyl transferase)
• Alternatively TK (thymidine kinase deficient)
• Cell Line Cannot Survive In Selection Medium
• Aminopterin Inhibits “De novo Pathway”, “Salvage Pathway” Is Not
Possibe Due To HGPRT or TK Deficiency
• It Is Also Ig Deficient
• It can not secret any immunoglobulins
• Aminopterin (folic acid antagonist) Blocks De novo Pathway
• P3.653 cells die in the presence of aminopterin
• They cannot utilize the “salvage pathway” because they are HGPRT
deficient
P3.653 MYELOMA

12. Somatic cell fusion
-Electrofusion : cells are allowed to fuse with the application of an electric field
-Done by using PEG medium
-PEG stands for Poly Ethylene Glycol
The New Hybrid Cell Exhibits Properties Of Both Cell Types
Unlimited growth
Secretes monoclonal antibody
Or Secretes cytokines

16. Selection of hybrid cells
-HAT medium is used for the selection of hybrid cells
-HAT stands for Hypoxanthine Aminopterine Thymidine
Nucleotide synthesis is essential for cell survival
In HAT medium, aminopterine blocks the cellular synthesis of purines and
pyramidines from simple sugars (denovo pathway)
But cells can succeed by using hypoxanthine and thymidine present in the
medium by salvage pathway using the enzyme HGPRT

17. -B cells are HGPRT+ and can survive in the HAT medium, but they undergo normal cell death after
some division
- In hybridoma technology, the myeloma cells used are HGPRT deficient
-So these cells can’t survive in HAT medium as Aminopterine blocks the Denovo pathway
Hybrid cells has HGPRT enzyme from the B cell as well as they have the ability to multiply repeatedly
as myeloma cells
So only hybrid cells can survive in HAT medium
HOW HAT MEDIUM WORKS IN THE SELECTION OF HYBRID
CELLS?

20. IDENTIFICATION AND ISOLATION OF THE
HYBRIDOMA CELLS
• • The first screening technique used is ELISA
• -Done by incubating the hybridoma culture supernatant, secondary enzyme labeled
conjugate and chromogenic substrate
• -Formation of a coloured product indicates a positive hybridoma

21. 1.Diagnostic Applications.
A. Biochemical analysis.
B. Diagnostic
imaging(immunoscintigraphy).
2. Therapeutics
A. Direct Agent.
B. Targeting Agent
3. Protein purification
Others..
Eg.
Abzymes
APPLICATIONS
OF MONOCLONAL
ANTIBODIES

22. DIAGNOSTIC APPLICATIONS
A. Biochemical
Analysis
B.Diagnositic
imaging
Eg. HIV test
Generally used
Radioisotopes are Iodine
131, technetium 99, Indium
chloride 111.
1. Radiolabel
the specific
monoclonal
antibody.
2. Inject them
intravenously into
the patients.
3. Detection by
using
SPECT(Single
Photon Emission
computed
tomography).
Used in detection of cancer, Bacterial
infections , Heart attack etc.

23. Metastatic medullary Thyroid carcinoma .
Patient was injected with Anti-CEA bispecific antibody with
indium111, and immunoscintigaphy was performed after 4
days. Liver , Bone and Brain shows good uptake of labelled
Antibody which shows metastatic tumour.

24. Therapeutics
1.Direct 2.Targeting agents
Eg . As
immunosupres
ent in organ
transplants.
OKT3
monoclonal
antibody
specifically
directed
against CD3
antigen of T
lymphocyte.
The toxins can
be coupled with
MAb’s to form
immunotoxins
.Anti –IL2-R
conjugated with
exotoxin of
Pseudomonas
sp. Can be used
to destroy
malignant T cells
of T cell leukemia
Some drugs
,
radioisotope
s can be also
attached to
MAb’s and
carried out
towards the
specific
tissue for
efficient
actions