Hybridoma technology involves fusing antibody-producing B lymphocytes with myeloma tumor cells to produce hybridoma cells. These hybridoma cells are immortal and divide indefinitely while continuously producing monoclonal antibodies of a single specificity. The key steps involve immunizing mice, fusing their spleen cells containing B lymphocytes with myeloma cells using polyethylene glycol, and selecting antibody-producing hybridomas by culturing the fused cells in HAT selective medium. This technique allows for the unlimited production of monoclonal antibodies that recognize a single antigen.

1. Hybridoma Technology
20 July 2021 Abhijit Debnath BP605T and Biotech Unit-1 1
CO3.1
Noida Institute of Engineering and Technology
(Pharmacy Institute) Greater Noida
Abhijit Debnath
Asst. Professor
NIET, Pharmacy
Institute
Unit: 3
Subject Name: Biotechnology
(BP605T)
Course Details
(B. Pharm 6th Sem)

2. Hybridoma
Technology
20 July 2021 Abhijit Debnath BP605T and Biotech Unit-1 2
 Production
 Purification
 Applications
 Blood Products &
 Plasma Substitute
CO1.1
Noida Institute of Engineering and Technology
(Pharmacy Institute) Greater Noida

3. 20 July 2021 Abhijit Debnath BP605T and Biotech Unit-3 3
• Hybridoma technology is one kind of Biotechnology.
• It is a hybridization technique which is used to produce antibody
producing hybrid cell.
• These hybrid cells are produced by fusing the genetic material of B-
lymphocyte with tumour cell.
• The antibodies produced are monoclonal antibodies as they are
all of a single specificity.
• Presence of B-lymphocyte genetic material helps in antibody
production, whereas capacity to divide indefinitely in the culture due
to the presence of tumour cell.
INTRODUCTION CO3.2

4. The production of monoclonal antibodies
was invented by Cesar Milstein and Georges
J.F
.Köhlerin 1975.
They pre-programmed B-lymphocytes to
respond to a single type of antigen or antigenic
determinant, therefore they produce single
type of antibody specific to the specific
antigen.
20 July 2021 Abhijit Debnath BP605T and Biotech Unit-3 4
HISTORY AND DEVELOPMENT OF HYBRIDOMA
TECHNOLOGY CO3.2

5. When an antigen reacts with B-lymphocyte receptors, lymphocytes divide rapidly and produce a clone
of B cells, all these B cells produce antibodies against that specific antigen and this is called as clonal
selection.
ThatisB-lymphocytesproduceonlyonetypeofantibodies whicharespecifictoonlyonetypeofantigen
orantigenicdeterminant. ButfullydifferentiatedantibodyproducingB-lymphocytecellsknown asplasma
cellsdoesnotdividewhenculturedinalaboratory.
The term hybridoma was coined by Leonard Herzenberg during his sabbatical in Cesar Milstein's
laboratoryin1976/1977.
20 July 2021 Abhijit Debnath BP605T and Biotech Unit-3 5
HISTORY AND DEVELOPMENT OF HYBRIDOMA
TECHNOLOGY CO3.2

6. 20 July 2021 Abhijit Debnath BP605T and Biotech Unit-3 6
• Mouse is immunized by giving antigen injection against which
monoclonal antibodies have to produced along with an
adjuvant. This is followed by booster doses of the antigen.
• After 72 hrs of immunization the spleen is collected from the
mouse.
• Then a free cell suspension medium of the spleen is
prepared using sterile serum free medium followed by
centrifugation.
• All these processes are applied for extraction of plasma cells
from the spleen.
Step-I CO3.2

7. 20 July 2021 Abhijit Debnath BP605T and Biotech Unit-3 7
• Maintain the plasma cells in the cell culture medium
for 16-24 hrs before fusion.
• It should be ensured that the cells are in early phase of
growth at the time of fusion.
• The myeloma cells are selected based on some criteria
like these cells themselves should not produce
antibodies and also they should contain a genetic
markers such as HGPRT(hypoxanthine-guanine
phosphoribosyltransferase) .
• This genetic marker helps in easy selection of the
resulting hybrid cells.
Step-II CO3.2

8. 20 July 2021 Abhijit Debnath BP605T and Biotech Unit-3 8
• At the time of fusion both myeloma & spleen cells are
counted & then mix in the appropriate ratio.
• Depending on the properties of the tumour cell, the mixing
ratio of spleen to tumour cell may vary from 5:1 to 2:1
• Following the mixing, the cells are centrifuged into a loose
pellet.
• The supernatant is removed & the pellet is mixed with 1 ml
of Polyethylene glycol(PEG) for 3 min.
• In doing so the pellet will be broken up into uniform small
clumps.
Step-III CO3.2

9. 20 July 2021 Abhijit Debnath BP605T and Biotech Unit-3 9
• Following the fusion, dilute the cells in serum free medium
slowly to reduce the osmotic disruption of the fused cell.
• Then centrifuge the cells & resuspend in HAT
(Hypoxanthine Aminopetrin Thymidine) medium.
• Then Hat medium containing the fused cells are allowed to
incubate in a CO2 incubator for 3-4 days.
Step-IV CO3.2

10. 20 July 2021 Abhijit Debnath BP605T and Biotech Unit-3 10
• This mixture of cell population is then cultured in selective
media known as HAT medium along with the drug
aminopterin.
• The HGPRT myeloma cells cannot divide in the HAT medium
due to the presence of aminopterin.
• The Specific antibody producing B-lymphocytes are unable
to divide continuously in the culture medium, therefore
eventually theydie.
Step-V CO3.2

11. 20 July 2021 Abhijit Debnath BP605T and Biotech Unit-3 11
• Only the hybridoma cells have got the ability to divide and
proliferate on the HAT medium because genome from the B-
lymphocyte makes them HGPRT positive and genome from
the myeloma cells they can divide indefinitely.
• Thus only the hybridoma cells or fused cells are selected
using selective media called as HAT medium.
Step-VI CO3.2

12. 20 July 2021 Abhijit Debnath BP605T and Biotech Unit-3 12
SUMMARY OF HYBRIDOMA TECHNOLOGY CO3.2