Monoclonal antibodies, following the development of this technology
in 1975, have become an extraordinarily important resource for medical
research, diagnosis, therapy, and basic science. In recognizing
the overwhelming importance of this technology to modern medical
science, it is also important to recognize that monoclonal antibody
production is still largely dependent on the use of experimental
In November 1997, The Office for Protection from Research Risks
(OPRR) at the National Institutes of Health, forwarded a letter
to all Public Health Service (PHS) awardee institutions and Institutional
Animal Care and Use Committees (IACUCs) on avoiding or minimizing
discomfort, distress, and pain in the care and use of animals for
the production of monoclonal antibodies using mouse ascites method
since there is evidence that the ascites method of monoclonal antibody
production causes discomfort, distress, or pain. Accordingly, the
IACUCs at all Public Health Service awardee institutions must critically
evaluate the proposed use of the ascites method. The Committee must
determine that a) the proposed use is scientifically justified,
b) methods that avoid or minimize discomfort, distress, and pain
(including in vitro methods) have been considered, and c) the latter
have been found unsuitable. Fulfillment of this three-part IACUC
responsibility, with appropriate documentation, is considered central
to an institution's compliance with its Animal Welfare Assurance
and the PHS Policy .
In keeping with the need to minimize both the use and the discomfort
and pain inflicted on experimental animals, the following considerations
and recommendations in the application of monoclonal antibody technology
Monoclonal antibody technology relies on experimental animals for
two basic steps. The first step involves immunizing mice. Although
there are alternatives to this step (either by in vitro immunization
steps or using recombinant libraries), these alternatives have not
proven to provide the same level of efficiency, specificity, or
affinity of antibody available from traditional immunization protocols.
In addition, with the advent of alternatives to Freund's adjuvant,
less than minimal or slight pain or discomfort accompanies this
step and therefore there is no compelling reason to consider alternatives
at this time. Careful consideration of the total number of required
animals should be the investigators main concern in justification
of this step .
A second common step that uses animals comes in antibody production.
In order to produce large quantities of monoclonal antibody, it
has been traditional to grow hybridoma cell lines as an ascites
tumor in vivo. In general, this procedure produces antibody titers
approximately 1000-10,000-fold higher than those obtained in tissue
culture. Since 1-10 ml of ascites fluid containing 1-5 mg/ml specific
antibody can be obtained per animal while hybridoma cells in culture
produce only 0.5-5 µg/ml, one mouse can produce antibody equivalent
to between 1 and 100 liters of tissue culture fluid. The main advantages
of ascites are the extremely high yield of antibody and that the
method is not excessively labor-intensive. However, its main disadvantage
is the potential pain and discomfort caused to animals, due to painful
peritonitis, abdominal tension, and infiltratively-growing tumors.
It should also be noted that the monoclonal antibody produced by
this method is contaminated by endogenous immunoglobulin and has
the potential for contamination by viruses or bioreactive cytokines
that may interfere with later use.
There are in vitro alternatives to monoclonal antibody production
by ascites. These include standard static or agitated suspension
cell cultures, membrane-based and matrix-based culture systems,
and high cell-density bioreactors. The disadvantages to these systems
include their substantially greater effort and higher labor costs,
increased costs due to the components of tissue culture media, and
the poor growth and/or antibody secretion of some hybridoma lines
in vitro. In particular, high cell-density bioreactors are probably
beyond the capability of most laboratories due to the high initial
and ongoing material and labor costs and the specialized expertise
In light of the above considerations, the New York University IACUC
requests that investigators consider the following recommendations
in designing monoclonal antibody production.
In general, in vitro methods for monoclonal antibody production
are considered standard and accepted practice. The use of the ascites
method requires rigorous and well-documented justification. Justifications
based solely on cost or convenience will not be considered adequate.
In addition, the NYU IACUC strongly urges the Medical Center to
establish a monoclonal antibody core facility with high-density
cell culture bioreactor capability that would obviate any need for
continued use of the ascites method.
- Most applications require only small quantities and low concentrations
of antibodies. Examples of common applications requiring only
small quantities of antibody include immunoblots, immunoprecipitations,
immunocytochemistry, flow cytometry, and small-scale affinity
columns. Such applications are easily accommodated by use of unpurified
tissue culture supernatants or by monoclonal antibodies purified
from tissue culture supernatants. For purposes requiring up to
approximately 10-50 mg of antibody, standard tissue culture methods
involving growth of up to 50 liters of hybridoma cells should
be considered the method of choice. Proposed use of ascites for
applications of this kind would require specific justification
(e.g., use of a hybridoma with unfavorable growth characteristics
in vitro) in addition to the considerations described in 3 (below).
- Small-scale membrane-based culture systems are available that
facilitate production of monoclonal antibodies in the range of
10-100 mg per culture in 5-30 days. These systems are relatively
inexpensive and do not require specialized facilities or expertise.
Investigators with applications that require up to 1 g of monoclonal
antibody should consider the use of this in vitro alternative
for production. Use of ascites production for such applications
would require specific justification demonstrating that special
circumstances warrant use of the in vivo method. In addition to
the considerations described in 3 (below), justification must
demonstrate that the disadvantages of ascites production (including
pain and discomfort to the animals and potential in vivo contamination)
are outweighed by the specific requirements of the individual
- Production of greater than 1 g of monoclonal antibody by in
vitro methods is probably outside the current capabilities of
most laboratories. For these applications, ascites production
is presently the only alternative for in-house production. Approval
of such projects will require adequate justification for the required
amounts of antibody . In addition, the investigator must demonstrate
the required expertise for working with tumor-bearing mice, including
an adequate daily monitoring system to insure that animals do
not experience unnecessary pain or discomfort. Assurance must
be made that no animal will be allowed to develop tumors larger
than 20% of host body weight. Ascites fluid must be harvested
on a single occasion only, either under terminal anesthesia or
post mortem. Animals must be killed without delay if they show
more than mild distress, overt tumor deposits or spread, or significant
dehydration or cachexia.
- The specific guidelines for consideration by Principal Investigators
when developing animal study proposals and for Animal Care and
Use Committees when reviewing proposals involving the mouse ascites
a. The volume of the priming agent should be reduced to as
small a volume as necessary to elicit the growth of ascitic
tumors and at the same time reduce the potential for distress
caused by the irritant properties of the priming agent. Although
0.5 ml Pristane has been standard for adult mice, 0.1-0.2 ml
has been found to be as effective for many hybridomas.
b. The time interval between priming and inoculation of hybridoma
cells as well as the number of cells in the inoculum are determined
empirically. Inocula range from 105 -107 cells in volumes of
0.1 - 0.5 ml and are usually administered 10 -14 days after
priming. Generally, very high concentrations are associated
with greater mortality and concentrations < 1 x 105 cells
elicit fewer ascitic tumors and these tend to have a smaller
volume yield. Cell suspensions should be prepared under sterile
conditions in physiological solutions.
c. Hybridomas should be MAP (mouse antibody production) or
PCR tested before introduction into the animal host to prevent
potential transmission of infectious agents from contaminated
cell lines into facility mouse colonies and possibly to humans
handling the animals.
d. Animals should be monitored at least once daily, seven days
a week by personnel familiar with clinical signs associated
with ascites production and circulatory shock.
e. Ascites pressure should be relieved before abdominal distension
is great enough to cause discomfort or interfere with normal
activity. Manual restraint or anesthesia may be used for tapping.
Aseptic technique should be used in withdrawing ascitic fluid.
The smallest needle possible that allows for good flow.
In accordance with the Animal Welfare Assurance and the Public
Health Service Policy, and to appropriately document that investigators
proposing the use of monoclonal antibodies have considered alternatives
to minimize discomfort, distress, and pain, in future please refer
to these recommendations before submitting protocols to the IACUC.
Additionally, subsequent to the November 1997 OPRR Letter, the IACUC
expects that investigators will incorporate these recommendations
into all new applications submitted for funding.