MONOCLONAL ANTIBODIES

K. PRAMEELA
Department of Biotechnology
GITAM UNIVERSITY
The Perfect World The Real World
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ANTIBODIES
STRUCTURE CLASS
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
mAb offer Reproducible,
Predictable & Potentially
inexhaustible supply of Ab
with exquisite specificity
Enable the development of
secure immunoassay systems.
NOT Powerful tools for
clinical diagnostic tests
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
PRODUCTION OF MONOCLONAL ANTIBODY
Step 1: - Immunization Of Mice & Selection Of Mouse
Donor For Generation Of Hybridoma cells
HYBRIDOMA TECHNOLOGY
ANTIGEN ( Intact cell/
Whole cell membrane/
micro-organisms ) +
ADJUVANT
(emulsification)
Ab titre reached in Serum
Spleen removed
(source of cells)
PRODUCTION OF MONOCLONAL ANTIBODY
Step 2: - Screening Of Mice For Antibody Production
HYBRIDOMA TECHNOLOGY
After several
weeks of
immunization
Serum Antibody Titre Determined
(Technique: - ELISA / Flow cytometery)
Titre too low
BOOST
(Pure antigen)
Titre High
BOOST
(Pure antigen)
2 weeks
PRODUCTION OF MONOCLONAL ANTIBODY
Step 3: - Preparation of Myeloma Cells
HYBRIDOMA TECHNOLOGY
Immortal Tumor Of Lymphocytes
+
8 - Azaguanine
Myeloma Cells

High Viability & Rapid Growth
HGPRT
-
Myeloma Cells

PRODUCTION OF MONOCLONAL ANTIBODY
Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells
&
Selection of Hybridoma Cells
HYBRIDOMA TECHNOLOGY
FUSION
PEG
MYELOMA CELLS
SPLEEN CELLS
HYBRIDOMA CELLS
ELISA PLATE
Feeder Cells
Growth Medium
HAT Medium
1. Plating of Cells in
HAT selective
Medium
2. Scanning of Viable
Hybridomas
PRODUCTION OF MONOCLONAL ANTIBODY
Step 4: - Cloning of Hybridoma Cell Lines by Limiting
Dilution or Expansion
HYBRIDOMA TECHNOLOGY
A. Clone Each +ve Culture
B. Test Each Supernatant for Antibodies
C. Expand +ve Clones
Mouse
Ascites
Method
Tissue
Culture
Method
EVOLUTION OF MONOCLONAL ANTIBODY
1. TRANSGENIC
DNA SPLICING / GENE KNOCK
OUT
2. LIBRARIES
a.BACTERIOPHAGE
b. mRNA
c. Cell Surface

ENGINNERED ANTIBODIES
Chimeric Monoclonal Abs
These Abs contain the variable region
from one source(mouse) and constant
region from other source( human)

Bispecific Monoclonal Abs
Heteroconjugates
One half of the antibody carries out
specific function and another half
other function
These can be constructed by
chemically

Applications of Monoclonal
Antibodies
Diagnostic Applications
Biosensors & Microarrays
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
APPLICATIONS
1. Detection of pregnancy
2. Diagnosing numerous pathogenic
microbes
3. Measuring the blood levels of
various drugs
4. Matching histocompatibility Ags
5. Detecting Ags of certain tumors
6. Radiolabelled Ags used in vivo for
detecting or locating tumor Ags



APPLICATIONS
Monoclonal Abs to breast cancer cells
is labeled with I
131
and introduced into
blood to detect spread of tumor to
regional lymph node
ABZYMES
1.Catalytic monoclonal Abs are
known as Abzymes.
2.They have the dual property of Ab
and enzyme
3.They are highly specific
4.They require less energy





ABZYMES
1.Protein cutting at specific A.A as
similar to R.E
2. Blocking viral infections
3. Dissolves blood clots in the targets
Eg : Plasmolysis
Streptokinase