Hybridoma technology

1. HYBRIDOMA
TECHNOLOGY
Ahmed Mahmoud Mayla
Department of Botany & Microbiology
Faculty of Science
Alexandria University

2. “…There is no biology without chemistry
and chemistry is the science of
informed matter. On the molecular
level, it represents storage while on
the supra-molecular level it
represents processing…” Jean-Marie
Lehn 1987 Nobel Laureate

3. Immune Response
• IMMUNOLOGY: is the study of IR
• Antigen: is the substance that when introduced to
the body, the body form IR (specific)
• Ag must be:
– MWt.
– Foreign(Non-self)
– Complex(glycoprotein more antigenic)
• Hapten + Protein can stimulate IR against both
• IR may be by Abs Humoral or by cells CMIR

4. CELLS OF THE IMMUNE SYSTEM
Bursa

6. ANTIBODIES
STRUCTURE CLASS

7. Antibodies are Proteins that Recognize Specific
Antigens

8. IMMUNOTHERAPY
Treatment of the disease by Inducing, Enhancing or
Suppressing the Immune System.
Active Immunotherapy: -
It stimulates the body’s own
immune system to fight the
disease.
Passive Immunotherapy: -
It does not rely on the body
to attack the disease,
instead they use the
immune system components
( such as antibodies)
created outside the body.

9. Antibody Therapy
Activation of B cells that recognize
different epitopes.
Immunization
Polyclonal antibody in serum
Isolate individual B cells
Monoclonal antibody
Ag

10. ANTIBODIES
Derived from different B
Lymphocytes cell lines
POLYCLONAL. MONOCLONAL.
Derived from a single B cell
clone
Enable the development of
secure immunoassay
systems.
NOT Powerful tools for
clinical diagnostic tests

12. PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Splenocytes
with PEG (polyethylene glycol)

13. HYBRIDOMA
Immunized mouse
Myeloma (mouse tumor derived from plasma cells)
Hybridoma
Fusion with PEG (polyethylene glycol)
Ig production
Ig secretion apparatus
Immortal
spleen
No Ig production
Ig secretion apparatus
Immortal
Ig production
Short-lived in
culture

14. Step 1: - Immunization Of Mice & Selection Of Mouse
Donor For Generation Of monoclonal cells
ANTIGEN ( Intact cell/
Whole cell membrane/
micro-organisms ) +
ADJUVANT
(emulsification)
Ab titre reached in Serum
Spleen removed
(source of cells)

15. Antibody Response After Exposure to
Antigen

16. Step 2: - Screening Of Mice For Antibody Production
After several
weeks of
immunization
Serum Antibody Titre Determined
(Technique: - ELISA / Flow
cytometery)

17. Step 3: - Preparation of Myeloma Cells
Immortal Tumor Of Lymphocytes
+ 8 - Azaguanine
Myeloma Cells
High Viability & Rapid Growth
HGPRT-
Myeloma Cells

18. Step 4: - Fusion of Myeloma Cells with Immune Spleen
Cells &
Selection of Hybridoma Cells
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

19. 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

20. EVOLUTION OF MONOCLONAL ANTIBODY
1. TRANSGENIC
DNA SPLICING / GENE KNOCK
OUT
2. LIBRARIES
a.BACTERIOPHAGE
b. mRNA
c. Cell Surface
HUMANIZATION

21. FDA APPROVED MONOCLONAL ANTIBODIES
H
2003
Psoriasis
Raptiva™
Genentech/Xoma
C
1997
Non-Hodgkin’s Lymphoma
Rituxan®
BiogenIdec/Genentech/Roche
M
2003
Non-Hodgkin’s Lymphoma
Bexxar®
Corixa/GlaxoSmithKline
C
2004
Colorectal Cancer
Erbitux ™
BMS/ImClone Systems
H
2003
Asthma
Xolair®
Novartis/Genentech/Tanox
PD
2002
Rheumatoid Arthritis
Humira™
Abbott/CAT
H
2001
Chronic Lymphocytic Leukemia
Campath®
Schering /ILEX Oncology
H
2000
Acute Myleoid Leukemia
Mylotarg™
Wyeth
C
1998
Acute Transplant Rejection
Simulect®
Novartis
C
1998
Crohn’s, Rheumatoid Arthritis
Remicade®
J & J
H
H
1998
2004
Breast Cancer
Colorectal Cancer
Herceptin®
Avastin ®
Genentech/Roche
H
1998
Viral Respiratory Disease
Synagis®
MedImmune/Abbott
H
1997
Acute Transplant Rejection
Zenapax®
PDLI
M
2002
Non-Hodgkin’s Lymphoma
Zevalin™
BiogenIdec
C
1994
Acute Cardiac Conditions
ReoPro®
J&J/Eli Lilly
M
1986
Organ Transplant Rejection
Orthoclone-OKT®
Ortho Biotech
Antibody
Type (2)
Date of FDA
Approval
Indications
Name of Product(1)
Company Name

22. 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