Preparation of Monoclonal Antibodies
In 1975 Kohler and Milstein reported the outcome
of fusing antibody-producing mouse spleen cells with
a mouse myeloma cell line. The monoclonal antibodies
produced were of the specificity dictated by the
spleen cells but in the quantity characterized by the
myeloma cell line. From this beginning the preparation
of monoclonal antibodies has become an important
tool in almost every scientific discipline. No longer
restricted to the immunology laboratory, monoclonal
antibodies are used in a variety of experimental
techniques and diagnostic assays and now, with
improvements in molecular engineering techniques,
are realizing their potential in therapeutic applications
When the need arises to produce monoclonal
antibodies to a particular antigen, the decision to
establish the technology, use commercial enterprises,
or approach private groups to produce them has to be
For laboratories that decide to establish the technology,
this article describes a method that has evolved
in our laboratory since the mid-1980s. The method is
robust, in routine use, and has produced hundreds of
monoclonal antibodies to a wide variety of antigens.
The method uses commercial high-quality reagents,
including cloning supplements that replace the messy
and wasteful use of in vivo
-derived feeder cells. The
reagents detailed are the ones used in our laboratory
but other manufacturers make equivalent reagents.
The intention of this article is to describe the
reagents, equipment, and techniques used to prepare
monoclonal antibodies. Full reviews of monoclonal
antibody production, including methods for characterization,
purification, use of monoclonal antibodies,
and antibody engineering applications, are available in
a number of monographs, including Campbell (1991),
Donohoe et al.
(1994) and Goding (1986).
Sterile culture technique and accurate record
keeping are essential for success in monoclonal antibody
preparation. The sterile culture technique is not
difficult to learn, with attention to detail paramount
for success. There are many excellent texts on basic
and advanced tissue culture techniques, including
Freshney (2002) and Harrison and Rae (2003).
II. MATERIALS AND
A. Essential Equipment
Sterile laminar flow hood, benchtop centrifuge,
liquid nitrogen storage or equivalent, 37°C water bath,
and a 37°C, 5% CO2
gassed tissue culture incubator. An
inverted microscope to view cell growth is also useful.
B. Cell Culture Reagents
RPMI 1640 (10 × 500ml) (Cat. No. 21870-092), penicillin
(10,000 U) / streptomycin (10,000 µg), L
(29.2 mg) (PSG×l00, Cat. No. 10378-016), 10 mM
sodium hypoxanthine, 40µM
thymidine (HAT×l00, Cat. No. 31062-011), 10mM
sodium hypoxanthine, and 1.6mM
(HT×l00, Cat. No. 11067-030) are from GIBCO Invitrogen
Corporation. Fetal bovine serum (FBS, Cat. No.
) is from JRH Biosciences; select batches of
FBS that have been screened for hybridoma production. Complete Freund's adjuvant (CFA, Cat. No.
77140) and incomplete Freund's adjuvant (IFA, Cat.
No. 77145) are from Pierce Biotechnology Inc.
Hybridoma fusion cloning supplement (HFCS, Cat.
No. 592247800) and polyethylene glycol 1500 in 75 mM
HEPES, sterile and fusion tested (PEG, Cat. No.
783641), are from Roche Diagnostics. Red blood cell
lysing buffer (8.3g/liter NH4
Cl in 0.01M Tris-HCl)
(Cat. No. R-7757) and trypan blue solution (Cat. No. T-
8154) are from Sigma. Dimethyl sulfoxide (DMSO, Cat.
No. 102950) is from Merck. ELISA-based isotyping kits
are available from a number of manufacturers, including
Roche Diagnostics (Cat. No. 1183117) and Pierce
Biotechnology Inc (Cat. No. 37502). Store all stock
reagents as recommended by the manufacturer.
C. Cell Culture Materials
Tissue culture plates, 24-well (Cat. No. 143982) and
96-well (Cat. No. 167008) flat-bottom plates with lids,
(Cat. No. 136196) and 80-cm2
(Cat. No. 178891)
tissue culture flasks, cryotubes (Cat. No. 375418),
10-ml disposable pipettes (Cat. No. 159633) and petri
dishes (Cat. No. 150350) are from Nalge Nunc Int.
V-bottom 30-ml tubes (Cat. No. 128A) and wide-bore
graduated transfer pipettes (Cat. No. PP88SA) are
from Bibby-Sterilin Ltd.
A container (Cat. No. 5100 Nalgene Nunc) or a foam
box with walls of about 1 cm thick is required for the
control rate freezing of cells.
In addition, we use Gilson Pipetman 5- to 20- and
20- to 200-µl pipettes and a Gilson Distriman repeating
pipette for cloning, all with sterile tips. Hamilton
500-µl glass luer lock syringes are used for immunization
(Cat. No. 1750 Hamilton, Reno. Nev). Sterile
forceps and scissors are also required.
D. Myeloma Cell Line
Several myeloma cell lines are commonly used
for fusion with murine spleen cells; most have been
derived from the P3X63Ag8.653 murine myeloma line
(Kearney et al.
, 1979). The myeloma clones SP2/0
(Shulman et al.
, 1978) and FO (de St Groth and
Scheidegger, 1980), derived from P3X63Ag8.65, are
commonly used. These cell lines are available from
the American Type Culture Collection (ATCC)
(http://www.atcc.org) as CRL1580, CRL1581, and
CRL1646, respectively. We use P3X63Ag8.653 cells that
have been maintained in our laboratory for many
years, which are screened regularly for mycoplasma,
and we select batches that have been previously successful
in hybridoma production.
The preparation of monoclonal antibodies depends
on a series of steps that require attention to detail and
careful laboratory management. The major requirement
is time; Table I gives an approximate time
frame for monoclonal antibody preparation. Phase I is
largely taken up by the immunization protocol, ensuring
that the screening method is working, growing
the myeloma cells, and finally performing the fusion.
Phase 2 encompasses testing, cloning, and freezing
and is more labor-intensive. However, the workload is
variable and unless the screening method is cumbersome
or many fusions are carried out, it is not a fulltime
In our experience the secret to success in monoclonal
antibody preparation is the screening method.
Ideally the method should be in place before the
animals are immunized. The method needs to be
robust and reliable and, as there may be 30 to 50
samples to screen a day, reagents and equipment need
to be readily available. The method should be appropriate
to the intended use of the monoclonal antibodies. Although many antibodies do perform in alternative
protocols, there is no guarantee unless they have
been screened appropriately. Some suggestions for
screening methods are included in Table II, but detailed
descriptions are outside the scope of this article.
B. Antigen Preparation and Immunization
A number of protocols that we have used to immunize
mice to a variety of antigens are shown in Table
II. Animals can make antibodies to a wide range of
molecular structures with two important general
exceptions: animals will not usually make antibodies
to self-antigen (see Section V) and will not usually recognize
small molecules. Small molecules need to be
conjugated to a carrier protein. Selection of the carrier
protein appears to be largely personal, but the ease of
and position of conjugation are important criteria.
When linking peptides to carrier proteins, Landsteiner's
principle (Landsteiner, 1945) should be taken
into account, that antibody specificity tends to be
directed to epitopes of the hapten furthest removed
from the functional group linked to the carrier protein.
Commonly used carrier proteins include keyhole
limpet hemocyanin (KLH) and bovine serum albumin
(BSA). If you are going to screen for monoclonal
antibodies by ELISA, make both peptide-KLH
and peptide-BSA combinations; immunize with one
and screen against the other. There are a number of
methods for the conjugation of peptides to carrier
proteins and the protocol selected will depend on the
amino acid sequence of the peptide and the epitope of
interest. However, if you are having a peptide synthesized,
consider adding a biotin tag; a streptavidinconjugated
carrier protein can then make a convenient
If high-affinity IgG monoclonal antibodies are
required, the immunization protocol needs to invoke
T-cell help. Hence, adjuvant and several booster injections may be needed and test bleeds should be
examined for the presence of antigen-specific IgG. If
IgG isotype monoclonal antibodies are essential, they
should be screened for specifically.
The protein antigen immunization schedule used in
our laboratory is shown in Table III. Most protocols
require antigen in adjuvant for immunization. Complete
Freund's adjuvant (CFA) is highly effective but
may cause toxic side effects. A number of new
adjuvants are becoming available, including Hunter's
TiterMax (http://www. titermax, com), Pierce' s
AdjuPrime Immune Modulator (http://www.
piercenet.com), and RIBI adjuvant systems
(http://www.corixa.com) among others. Advice from
the Institutional Animal Ethics Committee should be
taken on which adjuvants are recommended.
If CFA is used, it should only ever be used subcutaneously
for the primary immunization and Freund's
incomplete used for subsequent boosts.
Complete Freund's adjuvant causes inflammation
and granuloma formation in mice and will cause
severe injury in humans. Take suitable precautions to
prevent self-injection and employ eye protection.
Six-week-old female BALB/c mice are preferred for
immunization as the myeloma lines used for fusion
were derived from that strain. Routinely, we immunize
six animals, in batches of two, with a 2-week interval
between immunizing the next two animals.
Wear eye protection.
C. Test Bleeds
- Draw an equal volume of antigen in saline and CFA
into two separate Hamilton 500-µl glass luer lock
- Connect the syringes with a three-way stopcock and
emulsify the mixture slowly by passing material
from one syringe to the other.
- Check that an emulsion has been formed by taking
a drop of the mixture from the stopcock and placing
on water, an emulsion will float and not dissipate.
- Using the Hamilton syringe and a 21-gauge needle,
immunize the animals with 100µl of emulsion
subcutaneously at the base of the tail and nape of
Analysis of a test bleed can allow the fusion to be
postponed and the animals reimmunized if the antibody
titer is low or nonexistent. Test bleeds should
only be carried out by experienced personnel. Test
bleeds should preferably be from the lateral tail vein
using a 26-gauge needle and syringe. A maximum of
1% of the body weight of the animal should be collected,
about 100-200 µl. Larger volumes of blood can
be collected at the time of euthanasia and can provide
a valuable positive control sample for the screening
D. Myeloma Cell Preparation
About 1 week before the fusion, thaw a vial of the
myeloma cell line from the liquid nitrogen stock (see
Section IV, B).
The myeloma cells should be of known pedigree;
it is vital to maintain the myeloma line carefully.
Mycoplasma-contaminated stock or stock that has
been overgrown will result in poor production of
IV. CELL FUSION PROCEDURE
- Transfer the cells into 24-well plates at about 2× 105 per well, check cell numbers daily, and scale up as
they double in number into 25-cm2 flasks and then into
80-cm2 flasks containing 50ml of RF10.
- Maintain vigorous growth rates during the scaleup
period. The day prior to the fusion the myeloma
cells should be given a one-to-one split so that the cells
are in an exponential growth phase at the time of
- The number of myeloma cells required depends
on how many spleen cells are to be fused. Typically, we use a spleen to myeloma cell fusion ratio of 10:1.
Normal BALB/c mice produce about 108 leukocytes
per spleen, hence 107 myeloma cells are required for
- On the day of the fusion and before the animals
are euthanized, examine the myeloma cells carefully to
check that the culture is not contaminated.
A. Background Notes
- RFIO medium: To 500 ml of RPMI 1640 add 50 ml
of FBS and 5ml of PSG×l00. Store at 4°C for up to 14
days; after 14 days replace the PSG. Warm to 37°C in
a water bath before use.
- HAT selection medium: Reconstitute 100× lyophilized HAT supplement with 10ml of sterile distilled
water and store 1-ml aliquots at 4°C protected
from light. Add 1 ml HAT and 2ml of HFCS to 100ml
of RF10 medium. Make only as much as required for
the fusion. Warm to 37°C in a water bath before use.
- HT medium: To 500ml of RF10 medium add 5 ml
of HT supplement. Warm to 37°C in a water bath
- HT medium +2% HFCS: Add 2ml of HFCS to 100
ml HT medium. Warm to 37°C in a water bath before
HAT medium is used to selectively grow hybrids
following fusion. Aminopterin selects against unfused
myeloma cells and myeloma:myeloma-fused cells by
blocking the main synthetic pathway for DNA.
Unfused spleen cells do not have the capacity to
survive for more than a few days. In hybridomas,
hypoxanthine and thymidine supply purines and
pyrimidines for DNA synthesis via HGPRTase
salvage pathways derived from the spleen cells. The
HT medium acts as a rescue medium while the
aminopterin is being diluted out. Although the HT
medium can be withdrawn once the nucleoside
biosynthesis pathways are re-established, in our experience
it is rarely worth the effort.
Cells under pressure often require "supplements"
to maintain growth. In many monoclonal antibody
preparation methods, these are supplied by feeder
cells, usually mouse thymocytes or peritoneal washout
cells. In the method described, feeder cells are replaced
with hybridoma fusion cloning supplement HFCS,
which in addition to being preferred for reasons of animal ethics, is more convenient and, in our experience,
B. Fusion Protocol
- For each spleen to be fused, place a 1-ml aliquot
of PEG mixture and a 3- and a 7-ml aliquot of RF10 in
a 37°C water bath.
- After euthanasia, submerge the mouse in 70%
alcohol for a few minutes; this prevents hair and
bedding material from contaminating the laminar flow
- Place the animal on its right-hand side, grasp
the skin posterior to the rib cage with sterile forceps,
and make a small incision to cut the skin but not to
penetrate the subcutaneous fascia. With two pairs of
forceps, grasp the skin at either side of the incision and
pull. The skin should peel away, revealing the subcutaneous
fascia. Flush the area with 70% alcohol to
remove any stray hair. Using forceps and scissors, cut
through the subcutaneous fascia into the peritoneal
cavity and locate the spleen and remove by cutting
away the connective tissue.
- Place the spleen into a petri dish containing
about 15ml of RF10. At this point, blood can be
collected by opening the thoracic cavity, quickly
puncturing the heart, and collecting blood; this can
be useful as a positive control.
- Half-fill two 10-ml syringes with RF10 from the
petri dish and, using 21-gauge needles, gently disrupt
the spleen by injecting the medium. Inject the media
slowly, a little at a time; you should see clouds of cells
going into the medium and the spleen turning lighter
in color. Finally, when the spleen looks like a limp sack,
gently tease apart the spleen with the needle and flat
tweezers to remove any remaining cells. Discard the
spleen connective tissue.
- Gently pipette the cell suspension to break
up clumps and transfer into a 30-ml V-bottom tube,
ignoring large clumps and the remains of the spleen
connective tissue; let the tube stand for about 5 min.
- Remove cells from the tube and transfer into
another 30-ml V-bottom tube, leaving behind the debris
and connective tissue that has settled to the bottom.
- Pellet the spleen cells at 400g for 5 min.
- Resuspend the cell pellet in 5ml of red cell
lysing solution, leave for 5 min, and then fill the tube
with media and pellet at 400g for 5 min.
- Resuspend the spleen cells in 5 ml of media and
perform a cell count. The number of leucocytes
derived from a single spleen should be approximately
108 cells total.
- Wash and count the myeloma cells; for 108 spleen cells, 107 myeloma cells will be required.
- Add the myeloma cells to the spleen cell suspension
to give a spleen/myeloma cell ratio of 10:1.
Note: This is the critical bit, for the next 20min you will
be committed to fusing the cells.
- Pellet the cells (400g for 5 min) and remove all
of the supernatant; this is best done with a Pasteur
pipette attached to a vacuum line.
- Tap the pellet to loosen the cells.
- With a wide-bore transfer pipette, add 1 ml of
PEG solution per spleen. Gently mix with the pipette
for 10s and then continue to mix the cells gently by
tapping the tube for a further 50 s.
- Retrieve the 3-ml RF10 sample and slowly add
dropwise over a 10-min period continually mixing the
cells gently by tapping the tube. This can be accomplished
(for a right-handed person) by holding the
tube in the left hand between thumb and forefinger,
tapping the tube with the second or third finger,
depending on comfort and reach, and adding the
medium with a transfer pipette held in the right
hand. With a little practice a steady rhythm can be
- After the first 3 ml, retrieve the 7-ml aliquot and
add over the next 10-min period.
- Pellet the cells at 400g for 5 min and resuspend
in about 10 ml of RF10; loosen the tube cap to allow for
CO2 transfer and place the cells in the tissue culture
incubator for about 1 h.
- Pellet the cells at 400g for 5 min.
- Gently resuspend the cell pellet in a small
volume of the HAT selection medium.
- Add this suspension back into HAT selection
medium; 50ml of HAT selection medium will be
needed for every 107 myeloma cells used in the fusion.
- Add about 1 ml of the cell suspension to each
well of the 24-well plates. This is the equivalent of
2 × 105 myeloma cells per well. We prefer plating
the fusion into 24-well plates, as evaporation is less of
a problem. The larger volume also provides enough
supernatant for any screening assay.
- Label and place the plates in the tissue culture
With the exception of the 1-h incubation period,
the entire fusion protocol should take no more then 2
C. Maintaining Hybridomas
Examine the wells using the inverted microscope on
days following the fusion. Do not keep the plates out
of the incubator for more than about 10min, as media in the wells will cool and the pH may also change.
Large numbers of dead cells may be seen, this is
normal. You may also see some small moving particles;
this is due to Brownian movement of debris and does
not indicate bacterial infection.
After 7 days, media needs to be replenished, this is
also the beginning of diluting out the aminopterin
from the HAT selection medium. To each well add 1
ml of HT medium +2% HFCS. Add the medium slowly
so as not to break up any colonies; this is not critical,
but it is easier to judge when to test a well if you can
see the size of intact colonies.
From now on it is a matter of observing colony
growth and changes in the pH of the medium. Screen
for antibody from wells in which the medium is
yellow (acidic) and colonies are about 25% confluent.
Keep feeding wells on a 7-day cycle for slow
growers and as needed for fast growers. For the
second and subsequent feeds we use HT medium +1%
D. Testing and Cloning
When testing for antibody, ensure that appropriate
negative controls are used. A positive control can be a
dilution of the test bleed or of serum collected when
the spleen was removed. If screening by ELISA, falsepositive
"antiplastic responses" can be a nuisance. The
use of antigen-negative wells as a control can help
screen these out. It is a waste of time cloning cells from
wells that display an antiplastic response.
Cells from wells that are positive in the screening
assay need to be cloned and stored in liquid nitrogen
as soon as possible. Cloning is performed not only to
produce a monoclonal population, but also to stabilize
cell growth and eliminate antibody nonproducers.
Nonproducers often grow faster than cells producing
antibody and can quickly outgrow the antibodyproducing
We routinely clone at 3 cells per well and observe
about two-thirds of the wells with cell growth. This
approximates to Poisson's distribution of a probable
cell cloning number of 1 cell per well. If you find more
wells with cell growth, clone at a lower cell number
per well, 1 or even 0.5 cells per well.
- HT medium +2% HFCS: Add 2ml of HFCS to 100
ml of HT medium. Store at 4°C. Warm to 37°C in a
water bath before use.
- Trypan blue solution (Cat. No. T8154 Sigma)
- Gently resuspend the cells in a positive well of
the 24-well plate using a sterile transfer pipette; avoid
creating air bubbles. Transfer the cells to a new 24-well
- Perform a viable cell count at a 1:2 dilution in
trypan blue solution.
In this example we will work from a viable cell count
of 6 × 105 cells / ml
- To obtain 3 cells/well, perform a series of
dilutions: 1/1000 dilution = 600 cells/ml; add 10µl
cells into 10 ml of HT medium. From this make a 1/20
dilution = 30 cells/ml or 3 cells/100µl.
- Conveniently make this dilution by taking 500 µl
of cells from the 1/1000 dilution and place into 9.5 ml
of HT medium +2% HFCS. This gives 10ml of cells,
which is sufficient for one cloning plate at 100µl per
- Using a Gilson Distriman, add 100µl of cell suspension
to each well of a flat-bottomed 96-well plate.
Label the new plate with the date and code of the original
well and plate that it came from, e.g., P1D3 means
plate 1 row D well 3.
- Examine the cloning plates regularly and replenish
with HT medium +2% HFCS on at least day 7 but
beforehand if noticeable evaporation of media occurs.
Some methods suggest wrapping 96-well plates with
plastic film to slow down evaporation. This can be successful
but makes viewing the wells difficult and can
also give a false sense of security, care also needs to be
taken when removing the film so as not to contaminate
- Test wells that display cell growth as required,
reclone, and freeze cells from positive wells using the
same procedure. We clone a minimum of three times
in order to achieve monoclonality.
When storing positive wells in liquid nitrogen, we
routinely store two vials from the initial positive 24-
well plate. From subsequent 96-well cloning plates we
keep no more than three or four positive wells and
store about two cryotubes of each and the same at each
subsequent cloning step.
F. lsotyping and Scaling up of Hybridomas
Isotyping of the monoclonal antibody can be
performed with a commercial ELISA kit, this can help
confirm monoclonality and determine the strategy for
antibody purification from the culture supernatant.
Care should be taken in scaling up the cultures from
wells to flasks, ensure that cell growth is stable, and
split cells into a number of wells of the 24-well plate; we establish confluent growth in at least four wells
before transferring the cells to a 25-cm2
V. LIQUID NITROGEN STORAGE
The only means by which long-term survival of
hybridomas can be achieved is by storing cells in
liquid nitrogen. It is essential that an up-to-date
catalogue of stocks is maintained. Valuable hybridoma
stocks should be kept in more than one freezer,
preferably at separate sites.
A. Cell Freezing Protocol
- Solution A: Gently mix 10 ml of RF10 and 10 ml FBS.
This solution may be stored at 4°C indefinitely if
kept sterile. Warm to 37°C in a water bath before
- Solution B: Add 6ml of DMSO to 14ml of RF10. This
mixture causes an exothermic reaction and may be
stored at 4°C for several days if kept sterile. Warm
to 37°C in a water bath before use.
B. Cell Thawing Protocol
- Resuspend cells in solution A.
- To this add an equal volume of solution B. Mix the
suspension gently and dispense into labeled sterile
- After sealing the tubes, place them into a Nunc
freezing container or a foam box.
- Put the container into a -80°C freezer for 24 h, after
which the tubes should be transferred into a liquid
nitrogen store. The location and contents of the
tubes have to be catalogued carefully.
We strongly recommend that full-face protection be
worn when retrieving cells from liquid nitrogen.
- Remove vials using cryogloves and forceps.
- Transfer vials immediately to a shatter-proof
- Using a beaker, transfer warm water from the
37°C water bath into the container.
- The lid of the container need only be partially
removed to allow access with the beaker and replaced
as soon as water is added. The use of the shatter-proof
container prevents injury from tubes that explode on contact with warm water. This is a comparatively rare event but has
caused severe injury.
- After a few minutes when the contents have
thawed, remove and wash the vials in alcohol.
- Remove the cells with a transfer pipette and
place in a sterile 30-ml tube.
- Slowly add an equal volume of RF10 medium
- Let stand for 5 min.
- Add 10ml of RF10 and let stand for a further 5
- Centrifuge at 400g for 5min and resuspend in
HT medium for hybridomas and in RF10 for myeloma.
- We routinely start cells growing at about 2 × 105 cells/well in a 24-well plate. The addition of 1% HFCS
will speed up cell growth. Do not let cells overgrow
and scale up to other wells and flasks as required.
- The first priority is to maintain frozen stock, so
ensure that more tubes are stored in liquid nitrogen.
We have overcome tolerance to self-antigen by
linking the conserved peptide to a fusion protein and
using the construct as the immunogen. Using this
strategy, we have been successful in making antibodies
to a human peptide with 95% homology to the
murine protein (Cavill et al.
If purchasing a tissue culture incubator, ensure that
it can be cleaned easily, has readily accessible filters
that are changed easily, and does not have "hidden"
tubing that cannot be cleaned and acts as a harbor for
- Make sure that the marker pen ink used to label the
cryotubes tubes is resistant to alcohol.
- We cannot overemphasize the importance of storing
positive hybridomas in liquid nitrogen for subsequent
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