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Hybrimune Hybridoma Production System
Publication 015-1010191 Rev 4.0 • www.btxonline.com
Applications
There are three common uses of electrofusion.
• Hybridoma production as one step in monoclonal
antibody production
• Producing therapeutic hybrids for immunotherapy
• Nuclear transfer from one cell cytoplasm to another
cell cytoplasm
Hybridoma Production
One step in monoclonal antibody production is producing
hybridomas. The following are the basic concepts for making
antibody-producing hybridomas:
1. Standard myeloma cells are B-cell cancer cells that are
immortal. They have several important modifications.
One is that they have been made susceptible to being
killed by specific chemicals that do not kill B cells.
Another is that they generally do not produce antibody
on their own.
2. In humans and animals there are millions of different
types of B cells (lymphocytes) with different antibody
specificities. They are not immortal. B cells expand in
number in response to external events, stimulation by
antigen and T cells.
3. A hybridoma is produced by fusing an immortal B-cell
line (myeloma) that does not secrete antibody with
B-cells that have been extracted from blood. The fused
cells take on characteristics of both cells (the myeloma’s
immortality and the B cell’s specific antibody).
4. The resulting hybrid is cultured in a special medium that
kills unfused myeloma cells while unfused B cells die
naturally over time.
5. The hybrids are then placed in a 96-well plate with a
dilution where approximately one antibody secreting
hybridoma exists per well. After some growth, antibody
is detected in the culture supernatant. Cells from
positive wells are cloned.
A figure of merit is the number of productive wells with antibodies
and the number of cells required to give, on average, a positive
well. The number of functional, antibody-secreting, hybridomas is
less than the number of fusions.
Experiment
Number
Antigen Specific
E-Fusion
PEG
1
20
0
2
10
0
3
400
23
4
151
21
Mean
145
11
An example of E-fusion versus PEG was produced by M. Coccia,
Ph.D., Platform Development Group at Medarex, Inc., Milpitas,
CA. Transgenic human antibody-producing mice were used in
experiments comparing efficiencies of E-fusion to PEG fusion. Each
experiment used a portion of the same splenocyte preparation
from mice immunized with tetanus toxoid (TT) for comparison
of the E-fusion and PEG fusion methods. Results shown are the
number of TT antigen-specific clones generated by each method
normalized to the same number of cells.
These data are representative of additional experiments
(not shown) utilizing four different antigens. Taken together, all
experiments showed E-Fusion generated approximately ten-fold
more antigen-specific antibody clones relative to PEG fusion.
Figure 7 shows Cytospin™ prepared and Wrights-Giemsa
stained SP2/0 cells 30 minutes following large scale E-fusion. In
these E-fusion experiments more than 75% of cells in the final
population were fused with 2 to 3 nuclei per cell. Additional
Medarex data is presented in Appendix B.
Another process using human B cells is described in Li, et al.
PNAS
.
2006;103(10):3557-3562.
Figure 7: Medarex Fusion Example
Cell Electrofusion Tutorial