Hybridoma Technology

Hybridoma
A cell that has been engineered to produce a

desired antibody in large amounts, to produce

monoclonal antibodies.
It is produced in the laboratory from the fusion of an
antibody-producing lymphocyte and a nonantibodyproducing cancer cell, usually a myeloma or
lymphoma.

Metabolic Processes Involved

Monoclonal Antibody
an antibody produced by a single clone of cells or

cell line and consisting of identical antibody

molecules

Method
Step 1: Immunization of Mice and
Selection of Mouse Donors for
Generation of Hybridoma Cells
Step 2: Screening of Mice for
Antibody Production
Step 3: Fusion of Myeloma Cells
with Immune Spleen Cells
Step 4: Selection of Hybridoma
Cells
Step 5: Checking for Hybridoma
Cells
Step 6: Cloning of Hybridoma Cell
Lines

Step 1: Immunization of Mice and Selection of

Mouse Donors for Generation of Hybridoma Cells

Mice are immunized with

an antigen that is
prepared for injection
either by emulsifying the
antigen with Freund's
adjuvant or other
adjuvants.

Step 2: Screening of Mice for Antibody Production

After several weeks of

immunization, blood
samples are obtained
from mice for
measurement of serum
antibodies.

Step 2: Screening of Mice for Antibody Production

When the antibody titer is

high enough, the mice

are euthanized and their
spleens are removed for
in vitro hybridoma cell
production.

Step 3: Fusion of Myeloma Cells with Immune Spleen Cells

Spleen cells from the

immunized mouse are

fused with the previously
prepared myeloma cells.
Fusion is accomplished by
a technique called
somatic cell hybridization.

Step 4: Selection of hybridoma cells

The cells are then placed in

HAT (hypoxanthine,
aminopterin, thymidine)
medium.
Only the hybridoma cells that
have the ability to multiply
immortally and possess
HGPRT will survive in the HAT
medium

Step 5: Checking for hybridoma cells

The supernatants from each

culture are tested to find

those producing the desired
antibody. Since there may be
more than one hybridoma
cell in the original cultures,
single cells from each
antibody-positive culture
must be isolated and
subcultured.

Step 6: Cloning of Hybridoma Cell Lines

a. Inject the cells into the
peritoneal cavity of a
mouse, or;
b. use in vitro cell-culture
techniques

Sources:
https://www.youtube.com/watch?v=c_krTc9M1WU
http://www.globalresearchonline.net/volume1issue

2/Article%20017.pdf
http://www.microrao.com/micronotes/mcab.pdf