Monoclonal antibodies (mAbs) are homogeneous because they are pro-duced by hybrid cells derived from a single antigen-stimulated B cell. The production of mAbs involves two phases. In the first phase an animal (usually a mouse) is immunized with the antigen of interest. Immunization of the antigen is often performed with an adjuvant, as discussed above. Splenocytes are harvested from the responding animal, and are fused with a myeloma cell line for in vitro propagation.
Before the immunization protocol begins, the methodology for detecting the specific antibody of interest in the mouse sera and tissue culture supernatants is developed. Otherwise, significant time and animal resources may be wasted later in the mAb- developing phase. Test bleeds should be performed in order to determine if the mice are responding to the immunizations. Most immunologically based assays for determining if the desired antibodies are being produced require less than 10 microliters of mouse serum. Once an appropriate response has been confirmed the mice should be boosted again and typically after three days from the boost the mice should be euthanized and spleens harvested.
The second phase is production of adequate quantities of mAb for a project or analysis. There are two major methods: in vitro and the ascites method.
The ascites method has been one of the most popular means for producing large quantities of highly concentrated monoclonal antibodies since its inception in 1972. However, improved techniques and culture media have demonstrated that mAbs can be produced by in vitro techniques at a quality and concentration that are similar to that of ascites. The National Research Council’s report on Monoclonal Antibody Production specifically states “in vitro methods for the production of monoclonal antibodies should be adopted as a routine method unless there is a clear reason why they cannot be used…”. In accordance with the PHSPolicy and the Guide, alternatives to the use of animals (in vitro techniques) for the production of mAbs must be considered in place of the ascites method. (See the Office of Extramural Research Guidance concerning the Production of Monoclonal Antibodies in Animals, NIH Guide for Grants and Contracts, Notice OD-00-019, 2/3/2000, and the 11/17/97 OPRR Dear Colleague letter on Production of mABs Using Mouse Ascites Method.)
The ascites method should only be used after in vitro failure of each cell line has been demonstrated, or other adequate justification is provided. Analysis of individual cell lines is necessary because the production performance of each hybridoma cell line grown in vitro is highly variable. Despite this variability, work performed by Petrie indicates that at least 90% of all hybridomas that are placed on in vitro production protocols will yield adequate amounts of high quality mAbs.
Several resources for the in vitro production of mAb are available. Some institutions have core facilities that may provide an in vitro mAb production service. The NIH also sponsors a national cell culture core facility (National Cell Culture Center, Minneapolis, MN; http://www.nccc.com/).
In Vitro mAb Production References Jackson, L.R., B.A. Trudel, and N.S. Lipman. 1999, Small Scale Monoclonal Antibody Production in Vitro; Methods and Resources. Lab Animal 28(3):38-50.
Alternatives in Monoclonal Antibody Production, 1998, The Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and National Center for Research Resources (NCRR) Workshop.
Schulhof, J. (ed). 1999. Small-Scale Monoclonal Antibody Production, special edition Lab Animal; Autumn 1999.
Monoclonal Antibody References Kohler G., and C. Milstein.1975. Continuous culture of fused cells secreting antibody of predefined specificity. Nature 256:495-497.
Heidel J. 1997. Monoclonal Antibody Production in Gas-permeable Tissue Culture Bags Using Serum free Media. Center for Alternative to Animal Testing: Alternatives in Monoclonal Antibody Production, 8:18-20.
Jackson L.R., L. J. Trudel, J.G. Fox, and N.S. Lipman. 1996. Evaluation of hollow fiber bioreactors as an alternative to murine ascites production for small scale monoclonal antibody production. J Immunol Methods 189: 217-231.
OPRR Reports, 11/17/97, Number 98-01 Animal Welfare, Production of Monoclonal Antibodies Using Mouse Ascites Method.
Peterson N., and J. Peavey. 1998. Practical applications of in vitro monoclonal antibody production. Contemporary Topics Lab Animal Science 37:61-66.
Monoclonal Antibody Production. 1999. National Academy Press, Washington D.C.
Jackson L., L. Trudel, J. Fox, and N. Lipman. 1999. Monoclonal antibody production in murine ascites. I. Clinical and pathologic features. Lab Animal Science 49:70-80.
NIH Guide for Grants and Contracts. Notice OD-00-019, 2/3/00.
Polyclonal Antibody References
NIH’s ARAC Guidelines: Recommendations for Consideration in the Research Use of Inflammatory Agents. Adopted by full Committee of the NIHARC on 8/13/86. Reapproved - 5/8/96.
Halliday, L.C. et al. 2000. Physiologic and Behavioral Assessment of Rabbits Immunized with Freund’s Complete Adjuvant. Contemporary Topics 39(5):8-13.
Jackson, J.R. and J.G. Fox. 1995. Institutional Policies and Guidelines on Adjuvants and Antibody Production. ILAR Journal 37(3):141-152.
Sigel, M.B., Y.N. Sinha and W.P. VanderLaan. 1983. Production of antibodies by inoculation into lymph nodes. Methods Enzymol 93:3-12.
C.3.c. Breeding Colonies
Investigators maintain breeding colonies for a variety of reasons. A breeding colony may be required for an established animal model because:
that animal model is not commercially available,
young animals have very specific age or weight requirements that cannot be fulfilled by a commercial breeding colony, or
physiological status of the mutant animal is too severely affected for it to survive shipment.
Investigators developing a new spontaneous or induced mutant animal model need to maintain their own breeding colony because there is no alternative source for this mutant. While trying to establish a breeding colony for a new mutant model, the investigator is also simultaneously working to determine phenotype, to identify affected physiological system(s), and define inheritance pattern.
To review standard operating procedures for breeding colonies, the IACUC will need information about colony management. Examples of necessary information include:
number of breeders and number of young per cage,
breeding system including number of females per male or continuous versus interrupted mating,
Large numbers of animals may be required to maintain a breeding colony. The exact number of animals can only be approximated because it is im-possible to predict in advance the exact number and sex of offspring. The estimated number of animals should clearly distinguish between:
young that cannot be used in experiments because they are of the wrong genotype or sex, and
animals that will be subject to experimental manipulations.
Colony management practices should be briefly described in the investiga-tor's animal protocol, and justification provided for departure from standard institutional practices.
Determining which animals to include in the estimated number of animals on an animal protocol can be challenging to the investigator and the IACUC in the absence of IACUC-developed guidelines. The estimated number of animals that are kept for breeding purposes and not subject to any experimental manipulations should be part of the animal protocol.
Studies involving genetic analysis are animal intensive. Genetic analysis can involve determining if a single gene has dominant or recessive inheri-tance, identifying different genes involved in a quantitative (polygenic) trait,
or fine mapping to determine chromosomal location of a mutant gene. It is possible for the investigator to estimate the number of animals required, but difficult for the IACUC to evaluate this estimate in the absence of experience.
Up to 1200 mice are required to map a single gene with recessive inheri-tance and full penetrance, and have adequate numbers of progeny for developmental studies, phenotyping and linkage analysis. This number assumes a breeding colony of 10 to 12 pair matings with a 6- to 8-month reproductive lifespan, around 90% productive matings, replacement of breeders, and no unusual mutant infertility or mortality.
Up to 1100 mice are required for quantitative trait loci analysis using analysis of F2 progeny. The number assumes small breeding colonies of two inbred parental strains (4 to 6 pairs) and two reciprocal F1 hybrids (2 to 4 pairs), no unusual infertility, replacement of breeders at 6- to 8-month intervals, and generation of between 500 and 1000 F2 mice for genotyping.
Up to 750 mice are required to construct a congenic strain using “speed'” congenic genotyping methods. This number assumes a breeding colony of 10 to 12 breeding pairs, replacement of breeders, and progeny for pheno-typing and genetic linkage. If the homozygous mutant does not breed and the congenic strain must be developed using intercross matings, the esti-mated number of mice increases to 1,200.
After founder transgenic or 'knock-out' mice have been identified, between 80 and 100 mice may be needed to maintain and characterize a line. The number assumes up to five breeder pairs per line, breeder replacement, no unusual infertility and adequate numbers of weanlings for genotyping and phenotyping characterization.
If a study requires fertilized one-cell eggs, embryos or fetuses, the protocol should indicate the number of eggs, embryos or fetuses that are required for proposed studies. The estimated number of experimental animals may be limited to the number of female animals that are mated and euthanized or surgically manipulated to collect the required eggs, embryos or fetuses. In this situation, males might be listed as breeders if they are not subject to any experimental manipulation.
If a suckling animal will be subject to any manipulation, such as thymec-tomy, toe clip or ear notch for identification, tail tip excision for genotyping, or behavioral tests, the estimated number of manipulated sucklings must be
included in the number of animals used. If suckling animals will be euthanized at or prior to weaning because they are the wrong genotype or sex for the experiment, then they may be included as animals held or euthanized but not subject to experimental manipulations.
One option is for the IACUC to request estimated animal numbers as follows:
Estimated number of weaned and adult animals
to be subject to experimental manipulations _________*
Estimated number of suckling animals to be
subject to experimental manipulations _________*
*Estimated numbers should be further subdivided based on invasiveness of procedures using institutional criteria. Estimated number of breeders held but not
subject to experimental manipulations _________*
Estimated number of suckling animals to be
euthanized at or prior to weaning, and not
subject to experimental manipulation __________*
In summary, the IACUC’s role for oversight regarding breeding colonies includes ensuring that the need for a breeding colony has been established based on scientific or animal welfare concerns, that the procedures used in the breeding colony are evaluated and approved by the IACUC on a regular basis (e.g., as part of the semiannual program review), that there is a mechanism for tracking animals, and that the standards of care and animal well-being for the animals in the breeding colony are consistent with the Guide.
References Beamer, W.G., Senior Staff Scientist, The Jackson Laboratory. Bar Harbor, ME. Personal communication.
Festing, M.F.W. 1987. Animal production and breeding methods. In The UFAW Handbook on the Care and Management of Laboratory Animals. 6th ed., pp. 18-34. T B Poole (ed). Churchill Livingstone Inc., New York.
Fox, R.R. and B.A. Witham (eds). 1997. Handbook on Genetically Standardized JX mice. 5th edition, pp. 43-44, 120-125. The Jackson Laboratory. Bar Harbor, ME.
C.3.d. Field Studies
Federal requirements and the Guide focus primarily on the care and use of laboratory animals in research facilities. The same guiding principles, however, apply to the use of vertebrate species in field studies.
Application of the requirements and guidelines often pose unique chal-lenges to the investigator and the IACUC because of the nature of field research. For example, field sites are often at a distance and may be remote, making it impractical for IACUC inspections. One solution is to re-quire the investigator to provide photos, videotapes or other information that can help the committee evaluate the use of animals. For some projects the committee can find a consultant near the field site to perform an inspection and report to the IACUC. If the studies fall under the AWRs, at least two members of the IACUC must conduct the site inspections. Other difficulties relate to the nature of the research and the populations to be studied, which may be unfamiliar to the IACUC.
Professional field biologists in organizations devoted to the study of fish, amphibians, reptiles, birds, and mammals have prepared guidelines for field work with these populations; these guidelines form a useful reference and can assist the investigator in planning, and the IACUC in reviewing, field research using vertebrate animals. The references at the end of this section cite such guidelines. Appendix E includes a list of professional societies and contact information. These organizations can assist by referring the IACUC to appropriate individuals and authorities.
There is a comprehensive set of laws intended to protect wild animal populations. Appendix E describes these laws and the manner in which they are implemented and enforced. Virtually all activities involving birds, for example, require permitting under the Migratory Bird Treaty Act, the Endangered Species Act, the Bald and Golden Eagle Protection Act, or other permitting requirements. The investigator must be able to assure the IACUC that all necessary federal and state permits have been or will be obtained before research begins.
The proposed study can be assessed by the IACUC in a manner similar to laboratory studies if the protocol prepared by the PI addresses the following relevant items: