Guidelines for Monoclonal Antibody Production in Mice
In vitro methods are to be used for the production of monoclonal antibodies (MAb) unlessthere are clear scientific reasons why they cannot be used or why their use would represent an
unreasonable barrier to obtaining the product.6 It is the responsibility of the Institute/Center(IC) Animal Care and Use Committees (ACUC) to determine whether animal use is required forscientific or regulatory reasons.6 Strong scientific justification should be provided by theinvestigator. Examples of scientific reasons for justification of the use of the ascites methodthat IC Animal Care and Use Committees may find acceptable are listed in Table 1. References
describing alternatives to in vivo methods of monoclonal antibody production are available.1,3,78 When the mouse ascites method for producing MAb is required, every reasonable effort
should be made to minimize pain or distress, including frequent observation of the animal,limiting the number of survival abdominal wall punctures, and prompt euthanasia if signs ofdistress appear.2
The specific guidelines for consideration by Principal Investigators when developing Animal
Study Proposals (ASPs) and for ACUCs when reviewing proposals involving the mouse ascites
method are:
1. Biological material and animal products such as cell lines, tissues, and tumors that are
introduced into research animals can harbor animal pathogens (e.g. mycoplasma,
ectromelia virus, lymphocytic choriomeningitis virus, mouse hepatitis virus and others)
which can then infect NIH animal colonies. Principal Investigators are responsible for
ensuring that the biologic materials used in their study will not endanger the health of
the animals used in their study, other animals housed in the animal facility, orpersonnel. Hybridomas must be tested with a PCR-based or species-specific antibody
production assay before introduction into an animal host unless otherwise approved by
the Animal Program Director (APD), or their designee - generally the IC Rodent Import
Officer (RIO) or the Facility Veterinarian. The APD or their designee must review andapprove the test results prior to the use of the hybridoma cells on an approved ASP
which includes the ascites production being considered. Hybridomas tested years ago
with antiquated or incomplete pathogen-target sets should be re-tested. Hybridomas
obtained from other investigators on campus must be approved by the APD or theirdesignee prior to use even if they have been previously used in animals at NIH. This isrequired because of variations in the health status between different facilities and even
within facilities from room to room.
2. Any rodent-derived product, not just hybridomas, introduced into animals must beapproved on the Animal Study Proposal (ASP), known to be specific pathogen free, andif imported from a non-NIH source other than an approved commercial vendor, must beconsistent with NIH Policy Manual 3043-1 Introduction of Rodents, Rodent Products
and Rodent Pathogens from Non-Approved Sources. Laboratory personnel must always
1

be aware of any laboratory practices that could potentially transmit an infectious agent
into an animal facility. For example: antibodies/serum must be approved free ofinfectious agents if they are to be injected into animals or used in vitro to treat cells thatwill be transferred in vivo.
3. The generation of ascites fluid requires the use of a “priming” agent. A common“priming” agent used is Pristane, however, Freund’s Incomplete Adjuvant (FIA) has also
been shown to be an effective priming agent. Concern has been expressed about thepotential for discomfort and distress that may be associated with “priming” agents,
particularly Pristane.6 Due to this concern, many guidelines suggest a lower 0.1 to 0.2 ml
dose of Pristane. 2,3,5,6,9 It is also recognized that, as an ILAR report states, ‘in somestrains of mice, 0.2 ml might not be sufficient to produce ascites and that as much as 0.5
ml might be required.”6 A maximum dose of 0.3mls is recommended for FIA.3
Consideration for using the lowest doses of priming agents is strongly encouraged.
4. Cell suspensions must be prepared according to the ARAC Guidelines for Use of
Preservative-Free Pharmaceuticals and Parenteral Fluids in Laboratory Animals. The
number of hybridoma cells inoculated and the inoculum volume commonly range from105 -107 cells in volumes of 0.1 - 0.5 ml respectively, but may vary with the hybridoma
line.5 The standard time interval between priming and inoculation of hybridoma cells is
10-14 days. Generally, high cell numbers are associated with greater morbidity and lessthan 1 x 105 cells may elicit fewer ascitic tumors.
5. Once hybridoma cells are injected, animals should be monitored at least once daily,
seven days a week by personnel familiar with clinical signs associated with ascites
production.
6. Ascites pressure should be relieved before abdominal distension is great enough tocause distress or interfere with normal activity. Manual restraint or anesthesia may be
used for abdominal paracentesis (peritoneocentesis or ‘tapping’). The tap should be
performed by trained personnel using proper aseptic technique. The smallest needle
possible that allows for good flow (18-22 gauge) should be used. Fluid should be
removed slowly and allowed to drip from the hub of the needle rather than be aspirated
with a syringe. 4,6
7. Animal(s) should be monitored frequently over several hours following the tap toobserve for complications including signs of shock due to fluid withdrawal. Preemptive
fluid replacement with 1 – 2mls warm sterile saline administered subcutaneously before
the tap or intraperitoneally immediately following the tap may alleviate somecomplications especially in animals where more than a single tap will be performed.
Animals should continue to be monitored at least once daily, seven days a week by2

personnel familiar with clinical signs associated with ascites production, circulatory
shock, pain, and distress. Pale eyes, ears and muzzle and breathing difficulties areindicative of circulatory shock. Signs of pain or distress may also include hunched
posture, rough hair coat, reduced food consumption, loss of body condition, emaciation,inactivity, difficulty in ambulation, respiratory problems, and palpable solid tumor
growth. Animals should be euthanized promptly if there is evidence of hemorrhagicparacentesis, shock, pain, or distress.
8. The number of taps should be limited, based on the condition of the animal, and havejustification and ACUC approval. A maximum of three taps are recommended.

Table 1
Examples of scientific reasons for the use of the ascites method.6a.
b.
c.
d.
e.
f.
g.

h.
i.

The hybridoma cell line will not adapt well to in vitro conditions.
In applications where several different mouse MAb at high concentrations are requiredfor injection into mice, the in vitro method can be inefficient.
MAb from mouse ascitic fluids might be essential for experiments in which MAb are usedin vivo in mice.
Rat hybridoma cell lines do not generate ascites efficiently in rats, and usually adapt
poorly to in vitro conditions, but usually generate ascites in immunocompromised mice.
Downstream purification can lead to protein denaturation and decreased antibodyactivity.
Serum-free or low-serum conditions cannot provide sufficient amounts of MAb for some
purposes, such as the evaluation of new vaccines against infectious organisms.
Culture methods sometimes yield populations of IgG MAb that are glycosylated atpositions different from those harvested from mouse ascites fluid, thereby influencing
antigen-binding capacity and important biologic functions.
When hybridoma cells producing MAb are contaminated with infectious agents, such as
yeasts or fungi, the cells often must be passed through mice.
Some cell lines that do adapt to tissue-culture conditions become unable to maintainadequate production of Mab.

3References
1. Animal Welfare Information Center “Information Resources for Adjuvants and Antibody
Production:Comparisons
andAlternativeTechnologies
1990-1998”.
http://pubs.nal.usda.gov/information-resources-adjuvants-and-antibody-productioncomparisons-and-alternative-technologies
2. Behavioral, Clinical, and Physiological Analysis of Mice Used for Ascites Monoclonal
Antibody Production. Norman C. Peterson. Comparative Medicine 50(5): 516-526, 2000.
3. Canadian Council on Animal Care Guidelines on: Antibody Production. 2002.
http://www.ccac.ca/Documents/Standards/Guidelines/Antibody_production.pdf
4. Howard, Gary and Kaser, Matthew. Making and Using Antibodies: A Practical Handbook,Second Edition. New York, NY: CRC Press, 2014. p. 128.
5. ILAR Journal Volume 37, Number 3, 141-152, 1995.
6. ILAR report on Monoclonal Antibody Production. A Report of the Committee onMethods of Producing Monoclonal Antibodies. Institute for Laboratory Animal Research,
National Research Council. 1999. https://grants.nih.gov/grants/policy/antibodies.pdf
7. Johns Hopkins Center for Alternatives to Animal Testing. http://altweb.jhsph.edu/8. National Cell Culture Center. 2005. http://www.nccc.com
9. ILAR Journal Volume 46, Number 3, 227-320, 2005.

Initial ARAC approval – 06/12/96
Revised - 03/11/98, 03/27/02, 05/12/04, 09/12/07, 05/12/10, 03/13/13, 02/10/16
Archived – 1/23/20194