standard operating procedure - princeton …s/sop-monoclonal... · standard♦operating♦procedure...

STANDARD♦♦♦♦ OPERATING♦♦♦♦ PROCEDURE

PRINCETON UNIVERSITY

Written By: Date:

Approved By: Date: of 4

Section: IACUC SOP#: PRN – 3.2 (Ver. 8-2001)

TITLE: Production of Monoclonal Antibodies

PROCEDURES:

1) Species Choice: Fusion of mouse myeloma cells with mouse or rat spleencells yield a stable source of mouse and rat monoclonal antibodies.However, identification of some surface molecules can be hampered bythe evolutionary proximity of the rat and the mouse. Hamsters, which areevolutionarily distant form the mouse, are used for antibody productiononly upon failure to elicit a response in a mouse system. Metley, J.P.,Witmer-Pack, M.D., Aggar, R., Crowley, M.T., Lawless, D., Steinman,R.M. The District Leukocyte Integrins of Mouse Spleen Dendritic Cells asIdentified with New Hamster Monoclonal Antibodies. J. EXP. MED. (May1990) Vol. 171, 1753-1771.

Normally, two female Balb/c mice approximately 4 weeks old are used foreach experiment.

2) Immunization: The animal is held by grasping the loose skin over theshoulders with the thumb and forefinger, and holding the base of the tailwith the little finger, belly upwards. A 25-G needle and syringe is insertedinto the peritoneal cavity to a depth of 5mm, to one side of the midline,between the lower two nipples. The innoculum is injected slowly with abrief pause before the needle is withdrawn and the animal is returned tothe cage.

Mice, rats and hamsters are immunized 3-6 times at 2-3 week intervalsusing 200-300 microliter of an aqueous non-infectious antigen mixed50/50 with RIBI Adjuvant. (RIBI Immunochem Research, Inc., Hamilton,Montana, 59840) Rubach, J.A., Johnson, D.A., and Ulrich, J.T. (1995)RIBI Adjuvants: chemistry, biology and utility in vaccines for human andveterinary medicine, in ADJUVANTS: THEORY AND PRACTICALAPPLICATIONS (Stewart-Tull, D.E.S. ed.), Wiley, N.Y., pp.287-313.



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3) Retro-Orbital Bleeding: The animal is restrained by the scruff of the neckbetween the thumb and index finger. The tail is gently held against thebenchtop in such a way that the eye pointing up slightly bulges. A sterile100 microliter capillary tube (outer diameter less than 1 mm) is carefullyinserted into the cavity behind the mouse's eye, slight pressure is appliedand the capillary gently rotated until the sinus is ruptured and blood fillsthe tube (less than 5 seconds). The capillary is removed, the edge of theeye gently swabbed and the animal returned to the cage. Animals largerthan mice will be lightly anesthetized with Halothane prior to procedure.

Mice, rats or hamsters are normally bled once during an experiment atwhich time 100-200 microliter of blood is drawn. On rare occasions, if anadequate immune response is not detected after the first test bleed, onceadditional 100-200 microliter bleed is done from the opposite eye of theinitial bleed, at least three weeks after the initial bleed. The animal is thenkilled by CO2 asphyxiation and the spleen is removed. "It is possible totake up to 0.2 ml on repeated occasions from a narcotized living mouse(about 20 gm.), or approximately 0.7 ml of venous blood form the plexusretro-orbitals on a single occasion." J.H. Peters, H. Baumgarten (eds.)MONOCLONAL ANTIBODIES Springer-Verlag Berlin Heidelberg NewYork 1992.

As previous described, eye-bleeding is extremely rapid (2-3 seconds),relatively painless (similar to an injection), and does not result inpermanent injury to the eye or eye socket. Mice and rats can also bebleed form the tail vein by amputating a small portion of the tail or byincising the tail vein and collecting the droplets into a tube. This tailbleeding method allows no control over the amount of blood flow andfurthermore can result in an infection with the possible loss of the tailaltogether. Of course, hamsters cannot be tail bled because their tails aremere stubs. To take blood by cardiac puncture requires surgicalintervention performed by a highly trained technician and should becarried out only at the end of an experiment when the animal will be killed.It is my opinion that retro-orbital bleeding is the least painful/distressfulmethod of blood collection. Hamsters will be anesthetized with Halothaneprior to the procedure. MONOCLONAL ANTIBODIES Springer-VerlagBerlin Heidelberg New York 1992.



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4) Spleen Removal: The animal is killed by asphyxiation in CO2. The animalis then swabbed with 70% ethanol, the superficial skin pinched up over theleft side of the abdomen, and a small cut made over the spleen. The skinis torn back revealing the abdominal muscles, through which the spleenwill be visible. The abdominal wall over the spleen is pinched up withforceps, and a small incision made with fine scissors, taking care to avoidthe gut. The spleen is gently delivered through the incision, released bycutting its mesentery and placed into a sterile tube with growth medium.

5) Thymus Removal: The animal is killed by asphyxiation CO2 and swabbedwith 70% ethanol. The thymus is a bilobed, soft tissue which is ventraland just anterior to the heart. It is reached by first reflecting the skin andmuscle layer over the sternum. The ribs are then cut either side of thesternum, along most of its length. The thymus is grayish mass at the topof the thoracic cavity and is removed with fine scissors at its anterioraspect and placed in a sterile tube.

USE OF MOUSE ASCITES IN MONOCLONAL ANTIBODY PRODUCTION

General Statement:The use of mouse ascites in the production of monoclonal antibodies at

Princeton University is consistent with “The Report and Recommendations of theEuropean Center for the Validation of Alternative Methods (ECVAM) Workshop:Monoclonal Antibody Production” (11/96); the “Dear Colleague” letter written byOPRR (11/97); and the Princeton University IACUC letter regarding ascitesproduction in mice (3/98).

There are several, non-animal, alternatives to the use of mouse ascites.Wherever possible, these alternatives will be used. The Monoclonal AntibodyFacility has an excellent track record of producing many Mab’s using thesealternate techniques. However, the facility reserves the limited use of mouseascites for those antibodies demonstrated to be unamenable to alternativetechniques, and where large quantities of antibody are needed.

PROCEDURE:1) Priming of the abdomen peritoneum.

a) Adult female mice (at least 6 weeks old) are primed by injecting 0.5 ml ofincomplete Freund's adjuvant into the peritoneum.

2) Injection of hybridoma cellsa) 7 - 14 days after priming, 5 X 105 - 5 X 106 hybridoma cells are injected

intraperitoneally.



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3) Collection of abdominal fluida) First tap

i) The mouse, sedated with Halothane, is placed on its back, and an 18 -gauge needle attached to a 5 - ml syringe is inserted into theperitoneum at the animal's left side, with the needle pointing anteriorlyand with the point to one side of the spleen. The fluid is then carefullydrawn into the syringe. The mouse is then returned to its cage.

b) Second tapi) A second tap may be performed, if needed. The mouse, sedated with

Halothane, is placed on its back, and an 18 - gauge needle attached toa 5 - ml syringe is inserted into the peritoneum at the animal's left side,with the needle pointing anteriorly and with the point to one side of thespleen. The fluid is then carefully drawn into the syringe. The mouse ishumanely euthanatized by CO2 inhalation.

c) Euthanasiai) Euthanasia will be by CO2 inhalation.

SEARCH OF THE RELEVANT LITERATURE FOR ALTERNATIVEPROCEDURES WHICH MAY CAUSE PAIN OR DISTRESS, BASED ON THE3’Rs OF ANIMAL RESEARCH

General Statement:The literature search will be conducted annually for alternative procedures

and attached to this SOP. Each IACUC protocol will contain the written Narrative(see USDA Policy #12), however the database literature search will be within thisSOP.

Databases:NLM Gateway: (3 Full Databases)

(http://gateway.nlm.nih.gov/gw/Cmd)AltWeb: (http://www.altwebsearch.com/)

Keywords:Cell culture OR computer model* OR invertebrate? OR nonanimalAlternative? AND (refinement OR replacement OR reduction)Mice AND (Antibodies, Monoclonal)Mice AND AscitesMice AND “eye bleeding”.twMice AND “chemical peritonitis”twMice AND “peritoneal hybridoma?”.tw

PRINCETON UNIVERSITY Office of Research & Project Administration5 New South, Princeton, New Jersey, 08544-0036

TO: Research Community

FROM: Institutional Animal Care and Use Committee

DATE: August 30, 1999

SUBJECT: Ascites Production in Mouse

The Institutional Animal Care and Use Committee would like to inform investigators

who need to produce monoclonal antibodies that there is a new alternative to the injection of

antibody-secreting hybrid tumor cells into the peritoneum of mice for the production of very

large quantities of mABs. Although this has been a standard procedure for years, it is used less

and less because of the stress and pain it causes to the animal involved.

Novel and reliable technologies based on cell culture in hollow fiber cartridges yield very

large quantities of highly concentrated antibodies, in the range of 100 mg. at a concentration of

0.5 mg. per ml. A Unisys Bioreactor based on this technology can be used in the Monoclonal

Antibody Facility of the Department of Molecular Biology.

Another new technology for producing mAB’s is available from INTEGRA Biosciences

called CELLine. It is a novel membrane based compartmentalized disposable cell cultivation

system. This system yields antibody concentrations comparable to that of ascites (1 mg/ml.).

The INTEGRA Biosciences CELLine culture devices have no capital equipment costs, a low

purchase cost, provide concentrated product, reduce serum use, and reduce handling. All of

these attributes are available in a device as simple to use as a standard tissue culture flask.

For investigators who need only modest amounts of concentrated monoclonal antibody,

the collection of 1 liter of supernatant from conventional hybridoma cell cultures in excess of 10

mg. of antibody at the desired concentration.

In some particular cases, when very large quantities of antibodies are required and the hybridoma

cell does not grow well in a bioreactor, the production of ascites by injection of hybridoma cells

in the mouse peritoneum can be justified. Marty Marlow, at the Monoclonal Antibody Facility,

can help implement these techniques.

This Institutional Animal Care and Use Committee policy is consistent with

recommendations from the OPRR (Dear Colleague letter dated November 17, 1997), the

National Academy of Sciences ILAR Panel on Methods of Producing Monoclonal Antibodies,

and the European Center for the Validation of Alternative Methods report. Each of these reports,

and other reprints are available in the packet from the Princeton Institutional Animal Care and

Use Committee.

Please contact A. Bendelac or M. Weigert of the IACUC if you have any questions.

Results Summary

CategoryItemsFound

Actions

Journal Citations 25

Books / Serials / AVs 0

Consumer Health 0

Meeting Abstracts 0

Other Collections 0

Total 25

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Mice AND (antibodies, monoclonal)AND alternatives

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NLM Gateway Results Summary http://gateway.nlm.nih.gov/gw/Cmd?GMResultsSummary

Results Summary

CategoryItemsFound

Actions

Journal Citations 3


Consumer Health 0

Meeting Abstracts 0

Other Collections 0

Total 3

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Mice AND Ascites AND Alternatives

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Results Summary

CategoryItemsFound

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Journal Citations 40


Consumer Health 0

Meeting Abstracts 0

Other Collections 0

Total 40

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Mice AND "eye bleeding"

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Consumer Health 0

Meeting Abstracts 0

Other Collections 0

Total 1

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Mice AND "Ascites" AND "refinement"

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Results

Journal Citations ( 2 collections searched ) Displaying items 1 - 1 of 1 found

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Adenovirus-mediated gene transfer of herpes simplex virus thymidinekinase in an ascites model of human breast cancer.Yee D, McGuire SE, Brunner N, Kozelsky TW, Allred DC, Chen SH, WooSL.Hum Gene Ther. 1996 Jun 20;7(10):1251-7. PMID: 8793549 [PubMed - indexed for MEDLINE]From PubMed

Journal Citations ( 2 collections searched ) Displaying items 1 - 1 of 1 found

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Mice AND "Ascites" AND "refinement"

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CAAT: Techreport 8 Summary: The Johns Hopkins Center for Alternatives toAnimal Testing (CAAT) and the Office for Protection fromResearch Risks (OPRR) at the National Institutes of Health(NIH) offered a workshop on "Alternatives in MonoclonalAntibody Production" September, 1997,


Back Issues/Monthly Columns Summary: Dangerous Precedent Set in Animal Research?Legal Rights for All Great Apes? Does UK Opinion Predict USSentiments?

http://netvet.wustl.edu/species/birds/qb9415.txt Summary: : 47.8 Am33P Age-related changes in eggproduction, fertility, embryonic mortality, and hatchability incommercial turkey flocks. TPC, CO2 levels, NH3 levels, and airtemperature exhibited day-to-day variations but bird age effectwas not significant. Ma

Altweb: MAB Summary: Until the ARDF Workshop, what was missing weresimple, comprehensive, user-friendly guidelines to help IACUCsdeal with monoclonal antibody production protocols. TheGuidance Document, however, includes sufficient instructions

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Animal Models of Disease, QB QB 95-14 Summary: (Quick bibliography series ; 95-14) 1.Diseases--Animal models--Bibliography. 2. Animal models inresearch--Bibliography. Abstract: We investigated plasmalipoprotein profiles and the activities of tissue cholesterolregulating enzymes in Wistar fatty r

qb9419 Summary: insulin-dependent diabetes mellitus (Wistar fattyrat). increased in Wistar fatty rats compared with controls.Animal models for studies of relationships between diet anddiabetes.



The HSUS Pain & Distress Press Conference Summary: Public opinion surveys indicate strong concernabout pain and distress in laboratory animals. Table 1. Effect of

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