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Alternatives to Monoclonal Antibody Production (Proceedings)

IACUC Issues of In Vitro vs. In Vivo Monoclonal Antibody Production

Louis DeTolla, VMD, PhD
Director, Comparative Medicine Program
 Chief, UMAB Veterinary Resources
 The University of Maryland School of Medicine

The University of Maryland School of Medicine IACUC responsibilities in advising investigators on the use of appropriate methods of monoclonal antibody production focus on three primary issues: 1) the requirement of live animals for the production of monoclonal antibodies from hybridoma cells, 2) the practicality/efficiency of in vivo cultures in the production of the required amounts of monoclonal antibody and, 3) the potential for any rodent biologic (e.g. hybridoma cell line) to be contaminated by an adventitious pathogen when introduced into a healthy rodent that could lead to morbidity or mortality in that rodent or spread to other rodents in the facility and, by so doing, create a negative impact on specific biomedical research projects.

Our most current monoclonal antibody production guidelines state that 1) all hybridomas must be Mouse Antibody Production (MAP) tested for adventitious pathogens (e.g. murine viruses, mycoplasma) that could interfere with experiments or cause morbidity/mortality in individual rodents and/or place the animal facility at risk. All testing is provided by Veterinary Resources free of charge. 2) All investigators should produce monoclonals in tissue culture. Our facility provides tissue culture support in the production of monoclonals. This is provided as a fee-for-service activity and includes the production of monoclonal supernatants in standard or serum-free media, roller bottle culture, or hollow-fiber cultures for larger quantities of antibodies. 3) Ascites production is used as an alternative procedure if tissue culture methods cannot be used or are inadequate. IACUC approval is required.

In the event that ascites production is approved, our guidelines recommend: 1) pristane priming - .2 cc I.P. (do not use CFA), 2) mice must be observed from the onset of ascites (enlarged abdomen) and tapped within 24 hours, and 3) the first tap usually yields the most antibody (use 3 mice per hybridoma). Euthanize the mouse (e.g. CO2 overdose) just prior to the tap or sedate, tap and then follow with euthanasia. 4) Any mice found moribund or in respiratory distress should be euthanized immediately. 5) Cardiac puncture can also be performed under deep anesthesia in addition to the I.P. tap just prior to euthanasia.

It is essential that all hybridomas (as is the case for other rodent biologics to be used in vivo in rodents) be MAP-tested to ensure that the hybridoma is free of all adventitious pathogens that could represent a risk to the animal facility.

MAP testing involves culture of the cell line or hybridoma, harvest of confluent monolayers, and resuspension in media at a concentration of 108 cells/ml. Approximately 2-3 ml of the cell suspension is homogenized and inoculated into 3 to 5 five mice (.5 ml per injection) by multiple routes (IP, SC, IV). The mice are euthanized at 3-4 weeks and examined for gross and microscopic histopathology. Serum is collected for immunoassay which is run against a panel of agents including: Minute Virus of Mice (MVM), Mouse Hepatitis Virus (MHV), Reo Virus Type 3 (REO-3), Ectromelia Virus, Lymphocytic Choriomeningitis Virus (LCM), Mouse Rotavirus (EDIM), Mycoplasma pulmonis, Mycoplasma arthriditis, and Polyoma.

Cell Line Transmitted Agents Infecting Mice

AgentSignificanceResearch
Minute Virus of Mice
(MVM)
ModerateImmunosuppression
Mouse Hepatitis Virus
(MHV)
SevereImmunosuppression
Morbidity/Mortality
Reo Virus Type 3
(REO-3)
ModerateCell line contaminant
Morbidity/Mortality
Ectromelia VirusSevereMorbidity/Mortality
Lymphocytic Choriomeningitis Virus
(LCM)
SevereImmunosuppression
Morbidity/Mortality
Zoonotic
Mouse Rotavirus
(EDIM)
ModerateImmunocompromise
Morbidity/Mortality
Mycoplasma pulmonis
Mycoplasma arthriditis
SevereImmunomodulation
Respiratory illness
Morbidity/Mortality
PolyomaModerateTumors/Organ pathology
Cell line contaminant

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