Ascites Production (1)
2
1 NIH D irector’s letter, 12/10/99, http:// grants.nih.gov/grants/olaw/references/resp12109 9.pdf Guidelines for Ascites Production in Mice 1. Tissue-cultur e methods for the pro duction of monoclonal antibodies (M Ab) are th e default method unless there are clear scientific reasons why they cannot be used or why their use would represent an unreasonable barrier to obtaining the product. 1 2. When the mous e ascite s method for producing MAb is used, every r easonable effort should be made to minimize pain or distress, including frequ ent observation, limiting the number of taps [i.e. peritoneocente sis], and prompt euthanasia if signs of distress appear. 1 3. The specific g uideline s for cons ideration by Principal Investigators when developing animal study proposals and for Animal Care and Use Committees when reviewing proposals involving the mouse ascites method are: a. The volume of the pri ming agent s hould 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 distres s caused by t he 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 interv al between pr iming and ino culation of hy bridoma cells as well as the n umber of cells in the inoculum are determi ned empirically. Inocula range from 10 5 -10 7 cells in volumes of 0.1 - 0.5 ml and are usually administe red 10 -14 days after priming. Generally, very high concentr ations are associated with greater mortality and concentrations < 1 x 10 5 cells elicit fewer ascitic tumors and these tend to hav e a smaller volume yield. Cell suspensions should be prepared under sterile conditions in physiological solutions. c. Hybridoma s shou ld be MAP (mouse a ntibody prod uction) 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. Anima ls shoul d be mon itored at l east onc e daily , seven day s a week by personnel familiar with clinical signs assoc iated with ascites produc tion and circulatory shock. e. Ascit es press ure should be relie ved before a bdominal distension is great enough to cause discomfo rt or interfere with normal activity. Manual restrain t or anesthesia may be used for tapping. Aseptic technique should be used in withdr awing ascitic fluid. The smallest needle possible that allows for good flow
-
Upload
prateek-gupta -
Category
Documents
-
view
215 -
download
0
Transcript of Ascites Production (1)
1 NIH D irector’s letter, 12/10/99, http://grants.nih.gov/grants/olaw/references/resp121099.pdf
Guidelines for Ascites Production in Mice
1. Tissue-culture methods for the production of monoclonal antibodies (MAb) are thedefault method unless there are clear scientific reasons why they cannot be used or why their use would represent an unreasonable barrier to obtaining the product. 1
2. When the mouse ascites method for producing MAb is used, every reasonable effortshould be made to minimize pain or distress, including frequent observation, limitingthe number of taps [i.e. peritoneocentesis], and prompt euthanasia if signs of distress appear.1
3. The specific guidelines for consideration by Principal Investigators when developinganimal study proposals and for Animal Care and Use Committees when reviewingproposals involving the mouse ascites method are:
a. The volume of the priming agent should be reduced to as small a volume asnecessary to elicit the growth of ascitic tumors and at the same time reduce thepotential 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 beenfound to be as effective for many hybridomas.
b. The time interval between priming and inoculation of hybridoma cells as well asthe number of cells in the inoculum are determined empirically. Inocula rangefrom 105 -107 cells in volumes of 0.1 - 0.5 ml and are usually administered 10 -14days after priming. Generally, very high concentrations are associated withgreater mortality and concentrations < 1 x 10 5 cells elicit fewer ascitic tumors andthese tend to have a smaller volume yield. Cell suspensions should be preparedunder sterile conditions in physiological solutions.
c. Hybridomas should be MAP (mouse antibody production) or PCR tested beforeintroduction into the animal host to prevent potential transmission of infectiousagents from contaminated cell lines into facility mouse colonies and possibly tohumans handling the animals.
d. Animals should be monitored at least once daily, seven days a week bypersonnel familiar with clinical signs associated with ascites production andcirculatory shock.
e. Ascites pressure should be relieved before abdominal distension is great enoughto cause discomfort or interfere with normal activity. Manual restraint or anesthesia may be used for tapping. Aseptic technique should be used inwithdrawing ascitic fluid. The smallest needle possible that allows for good flow
should be used (18 -22 gauge).
f. Animal(s) should be monitored frequently over several hours following the tap toobserve possible signs of shock due to fluid withdrawal. Pale eyes, ears andmuzzle and breathing difficulties are indicative of circulatory shock. Shock maybe prevented or treated with 2 -3 ml warm saline or lactated ringers administeredsubcutaneously.
g. The number of taps should be limited, based on good body condition of theanimal. A maximum of three survival taps (the 4th being terminal) arerecommended. Additional taps should have individual ACUC approval.
h. Animals should be euthanatized appropriately before the final tap or promptly if there is evidence of debilitation, pain or distress. Signs of distress includehunched posture, rough hair coat, reduced food consumption, emaciation,inactivity, difficulty in ambulation, respiratory problems, and solid tumor growth.
References
1. Behavioral, Clinical, and Physiological Analysis of Mice Used for Ascites Monoclonal Antibody Production. Norman C. Peterson. Comparative Medicine 50(5): 516-526,2000.
2. ILAR Journal Volume 37, Number 3, 141-152, 1995.
3. ILAR report on Monoclonal Antibody Production. A Report of the Committee on Methods of Producing Monoclonal Antibodies. Institute for Laboratory Animal Research, NationalResearch Council. 1999. http://grants.nih.gov/grants/policy/antibodies.pdf
Approved by ARAC, June 12, 1996Revised - 3/11/98Revised - 3/27/02Revised - 5/12/04
Page 2
