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Hybrimune Hybridoma Production System
Publication 015-1010191 Rev 4.0 • www.btxonline.com
Hybridoma Production:
E-Fusion vs. PEG Medarex Results
The data here was produced by Marco Coccia, Ph.D. in the Platform
Development Group, Medarex, Inc., Milpitas, CA using the BTX
Hybrimune Commercial Electrofusion System. BTX has permission
from Medarex to release this information.
In preparation for the fusion, mice were immunized
intraperitoneally or subcutaneously every 2 to 4 weeks with an
antigen plus Ribi adjuvant. The mice were bled periodically to
determine if an adequate antibody titer had developed. Mice were
given an intravenous antigen boost 3 to 4 days before harvesting
spleens to activate B cells and increase the number of antigen
positive cells located in the spleen. Spleens were collected from
adequately immunized mice just prior to the electrofusion.
For the fusion, mouse spleen cells and SP2/O mouse myeloma
cells were washed twice in Cytofusion medium. The cells were at
a concentration of 10 million cells per ml. A volume of 4.5 ml of
each cell suspension was mixed then placed into a 9 ml volume
electrofusion chamber. Cells were aligned and fused using the
following parameters.
After electrofusion, cells were left in the fusion chamber for 30
minutes. Samples of cells from wells were then analyzed by Wrights
stain of Cytospin preparations of the samples. The bulk of the cells
were cultured in HAT medium. The cells were cultured in 96-well
plates at 5000 cells/ml.
Total clones were counted by screening the wells in 96-well plates
by eye for hybridoma growth on day 7 to 9. The number of clones
was mathematically calculated using a poison distribution analysis.
Wells were screened for presence of IgG antibody and antigen
specificity using ELISA or an automated fluorescent screening
system (HTRF). Data collected during the screening was normalized
to 100 million cells to allow direct comparison of differed fusion.
Symbols used:
g
The immunoglobulin heavy chain for IgG
antibody (includes IgG 1-3)
k
One of two immunoglobulin light chains
#
g
,k
The number of wells that contained IgG
antibody of any specificity (the number of
wells with hybridoma clones secreting IgG
antibody)
#Ag,
g
The number of wells with hybridoma clones
secreting IgG antibody with specificity for
the antigen of interest
Ag
antigen, the specific antigen used in the
screening
TT
Tetanus toxoid
The table shows the results of 12 experiments. For electrofusion,
the average number of wells in the 12 experiments that produced
IgG-secreting hybridomas was 542. Of those, 91 bound to the
antigen of interest. For the PEG fusions, the average number
of wells in the 12 experiments that produced IgG-secreting
hybridomas was 58 and 12 of those were antigen specific. This
means that on the average electrofusion produced approximately
9 times as many IgG producing hybridomas and 8 times as many
antigen specific hybridomas. However, there were many cases
where E-fusion produced results and PEG did not.
Pre-pulse Sine Wave
40 V to 60 V, 1.4 MHz for 15
seconds
Pulse
Amplitude 800 volts, width
40 ms, one pulse
Post pulse Sine Wave
60 V to 5 V, 1.4 MHz for 30
seconds
Appendix A: Medarex Data
Appendices