fusion: Immortalization of sensitized B lymphocytes
from immune mice. Overview We outline a simple and contemporary protocol for the
development of monoclonal antibodies using
hybridoma fusion in immune mice (1). While the basic
style of this fusion is similar to others (2,3), this protocol
has several subtle but significant modifications. These
include the use of: spleen perfusion rather than
crushing to separate the spleen cells (which reduces
the amount of contaminating fibroblast and lipoidal
material) ; commercial Hybridoma CLoning Factors
(rather than feeder layers); commercially prepared
semi-solid HAT containing agarose, rather than limiting
dilution. Elements of this protocol span several
research institutions and many years of experience
(4,5,6). Material Sterile 10, 25, and 50 ml serological pipettes, Pipet-Aid,
15 and 50 ml centrifuge tubes (Falcon sterile), Tissue
culture flasks (25 cm2, 75 cm2 and 125 cm2), indelible
waterproof marker. Sterile 1 ml pipette tips for Gilson
p1000 pipetteman.
370C waterbath and thermometer.
Humidified 370C, 5% CO2 tissue culture cabinet.
Class II Biological Safety Cabinet.
Inverted Microscope.
Benchtop centrifuge containing a 4 swinging bucket
rotor, at room temperature.
Stopwatch or timer.
Multichannel pipettor and appropriate sterile tips, sterile
disposable petri dishes. Sterile 96-well flat-bottomed
cell culture plates.
Reagents:
1-3 x 10 8-9 immune spleen cells
1-6 x10 7-8 myeloma cells in log phase of growth
Complete Media No Sera (CMNS) for washing of the
myeloma and spleen cells.
Hybridoma medium CM - HAT {Cell Mab (BD), 10%
FBS (or HS) ; 5% Origen HCF (hybridoma cloning
factor) containing 4mM L-glutamine and antibiotics} to
be used for plating hybridomas after the fusion.
Hybridoma medium CM - HT (NO AMINOPTERIN) {Cell
Mab (BD), 10% FBS 5% Origen HCF containing 4mM
L-glutamine and antibiotics} to be used for fusion
maintenance stored in the refrigerator at 4-60C. feeding
fusions on days 4, 8, and 12, and subsequent
passages.
Thawed inactivated and pre-filtered commercial Fetal
Bovine serum (FBS) or Horse Serum (HS) stored in the
refrigerator at 40C. Must be pretested for myeloma
growth from single cells.
L-glutamine, 200mM, 100X solution stored at -200C
freezer.
The L-gln is thawed and warmed until completely in
solution. The L-gln is dispensed into media to
supplement growth. L-gln is added to 2 mM for
myelomas, and 4 mM for hybridoma media.
Penicillin, Streptomycin, Amphotericin (antibacterial-
antifungal) is stored at -200C until needed and it is
thawed and added to Cell Mab Media to 1%.
Cell Mab Media, Quantum Yield from BD is stored in
the refrigerator at 40C in the dark. Myeloma growth
media is Cell Mab Media with added L-gln to 2 mM and
antibiotic/antimycotic solution to 1% and is called
CMNS. Do not add antibiotics to media when growing
myelomas for stock, only for pre-fusion growth. Indicate
presence or absence of each reagent on the bottle
label. Do not adjust the pH as it contains HEPES
biological buffer already.
1 bottle of PEG 1500 in Hepes (Roche) (use fresh bottle
that has never been opened)
8-Azaguanine is stored as the dried powder supplied
by SIGMA at -700C until needed. Reconstitute 1 vial /
500 ml of media and add entire contents to 500 ml
media (eg. 2 vials/ litre).
Myeloma Media is CM which has 10% FBS (or HS) and
8-Aza (1 X) stored in the refrigerator at 40c.
Clonal cell medium D (Stemcell, Vancouver) contains
HAT and methyl cellulose for semi-solid direct cloning
from the fusion. This comes in 90 ml bottles with a CoA
and must be melted at 37Oc in a waterbath in the
morning of the day of the fusion. Loosen the cap and
leave in CO2 incubator to sufficiently gas the medium D
and bring the pH down.
Hybridoma supplements HT [hypoxanthine, thymidine]
are to be used in medium for the section of hybridomas
and maintenace of hybridomas through the cloning
stages respectively.
Origen HCF can be obtained directly from Igen and is a
cell supernatant produced from a macrophage-like cell-
line. It can be thawed and aliqouted to 15 ml tubes at 5
ml per tube and stored frozen at -200C. Positive
Hybridomas are fed HCF through the first subcloning
and are gradually weaned. It is not necessary to
continue to supplement unless you have a particularly
difficult hybridoma clone. This and other additives have
been shown to be more effective in promoting new
hybridoma growth than conventional feeder layers.
Procedure At least one week prior to expected fusion date thaw a
fresh vial of myeloma cells. It is advisable to keep
several flasks at different densities so that you can
choose the best one on the day of the fusion. We
generally try to use a flask that is actively dividing and
at a cell density of 3-6x105 cells/ml. Do not let them
overgrow or they will enter a decline phase.
Two to five days before the scheduled fusion give a
final injection of ~5ug of antigen in PBS i.p. or
intravenously in tail vein of the mouse (with high titer
already determined).
1. Spin down myelomas and wash with 30 ml serum
free media (CMNS has glutamine). Use tabletop
centrifuge at 850 rpm for 12 minutes. Perform viable
cell count with trypan blue exclusion principle, and
wash cells with 30 ml of RPMI-CMNS. Spin down as
above, resuspend in CMNS and disperse. Leave at
37°C until spleens are retrieved.
Test aminopterin sensitivity. Keep 1 million myeloma
cells for control plate and transfer into a 15ml conical.
To do so, add 15 ml of HAT media to the million
myeloma cells and plate out 2 drops/well on a 96 well
plate.
2. Remove spleen from mouse in the biohazard facility.
Euthanise the mice and submerge it in 70% ETOH. Let
the mouse air dry on its right side on a paper towel.
Remove spleen using sterile instruments and carefully
put into labeled 10 ml of RPMI-CM with antibiotics and
20% FCS for transport back to the lab. Dispose of
mouse and leave facility.
3. Place spleen into sterile petri dishes. Add 10 ml of
RP-I-CMNS and perfuse the cells out of the spleen.
Poke the spleen 8-10 times with an 18 ga needle (hold
with sterile forceps). Use a 21 ga on a 3 ml syringe to
draw up some RPMI. Inject the RPMI slowly into the
spleen about 50-100 times until nearly all the cells are
washed out. Discard the spleens into the biohazards
bag.
4. Collect and transfer the spleen cells to a new 50 ml
conical tube. Rinse out the dish 2X with 10 ml of RPMI-
CMNS and pool with the first 10 ml (the use of perfusion
removes the production of large debris seen with
grinding, and obviates the need to let the debris settle).
Spin down at 900 rpm for 12 minutes. Discard the
supernatant to bleach container. Wash the cells with
another 30 ml RPMI-CMNS. Remove a small sample
and count the viable cell/ml and spin again as above.
Combine the cells at a ratio of 5:1 (spleen cells:
myeloma cells) and never 1X10 myeloma cells.
5. Wash both the myeloma and spleen cells 2 more
times with 30 ml of RPMI-CMNS. Spin at 800 rpm for
12 minutes.
6. Remove supernatant and resuspend cells in 5 ml of
RPMI-CMNS and pool together. Fill volume to 30 ml
and spin down as before.
7. Aspirate all fluid into bleach vessel. Break up pellet
by gently tapping on the flow hood surface. Add 1 ml of
BMB REG1500 (prewarmed to 37°C) dropwise with 1
cc needle over 1 minute. Swirl and tap the conical
gently while adding the PEG to resuspend the cells.
8. Add 1 ml of RPMI-CMNS to the PEG cells gently over
1 minute while swirling (to dilute the PEG).
9. Add 8 ml RPMI-CMNS over 2 minutes to slowly dilute
out the PEG.
10. Incubate the cells in the 37°C waterbath for 10
minutes. Centrifuge the cells at 700 rpm for 10 minutes
(the membranes are still very weak).
11. Aspirate all fluid, and add 5 ml of RPMI-CM-10%
FCS WITHOUT RESUSPENDING THE CELLS! The
cells will disperse adequately by simply adding the
media at this point.
12. Incubate @ 37°C another 10 minutes.
(12.5 Meanwhile put aside 1 ml of Clonacell medium D
for myeloma testing. )
13. Gently dilute cells in 5 ml of Complete media and
transfer into 95 ml of Clonacell Medium D (HAT) media
(with 5 ml of HCF) and plate out 10 ml per small petri
plate.
14. Dilute about 1000 P3X63 Ag8.653 myeloma cells
into 1 ml of mediu D and transfer into a single well of a
24 well plate. This is the myeloma/HAT control. P
15. Place plates in incubator two plates inside of a
large petri plate, with an additional petri plate full of
dH20 without a lid for humidity. Leave for 10-18 days
under 5% CO2 overlay at 37 degrees.
15. Pick clones from semisolid agarose into 96 well
plates containing 150-200 ul of CM- HT. Screen sups 4
days later in ELISA. Move positive clones up to 24 well
plates.
16. Heavy growth will require changing of the media at
day 8 (+/- 150 ml). Should see macroscopic colonies at
this time.
At this time can decrease the HCF to 0.5% (gradually-
2%, then 1%, then 0.5%) in the cloning plates.
17. Isotype via supernatants and grow up for ascites/
large flask production and further freeze down. Troubleshooting
I strongly reccommend to use Southern Biotech Goat
anti-Mouse Ig (H+L)HRP Chains for screening
supernatants. We have screened and developed Mabs
to various antigens and have surprisingly found out that
using some secondary reagents from several other
major companies can result in weak or no reactivity.
Keywords: monoclonal antibody ; hybridoma ; fusion
Reference 1) Kohler G, and C. Milstein Continuous cultures of
fused cells secreting antibody of predefined
specificity.1975. Nature 256: 495-497.
2) Lane, R.D. A short duration polyethylene glycol
fusion technique for increasing production of
monoclonal antibody-secreting hybridomas. 1985. J.
Immunol. Meth. 81:223-228.
3) Harlow, E. and D. Lane. Antibodies: A laboratory
manual.Cold Spring Harbour Laboratory Press. 1988.
4) Kubitz, D. The Scripps Research Institute. La Jolla.
Personal Communication.
5) Zhong, G., Berry, J.D., and Choukri, S. (1996)
Mapping epitopes of Chlamydia trachomatis
neutralizing monoclonal antibodies using phage
random peptide libraries. J. Indust. Microbiol. Biotech.
19, 71-76.
6) Berry, J.D. , Licea, A., Popkov, M., Cortez, X., Fuller,
R., Elia, M., Kerwin, L., and C.F. Barbas III. (2003)
Rapid monoclonal antibody generation via dendritic
cell targeting in vivo. Hybridoma and Hybridomics 22
(1), 23-31.
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