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UNIVERSITY OF SOUTHERN MAINE
Office of Research Integrity & Outreach
Procedure #:
Date Adopted:
Last Updated:
Prepared By:
Reviewed By:
Procedure Title:
03-014
6/16/2006
2/14/2012
Sarah Gilliam, Animal Facilities Manager
Ivy Bergquist, Asst. Manager of Animal Facilities
USM IACUC Committee
Guidelines for the Use of Adjuvants in Animals
1.0 Purpose: To insure that proper technique is used to limit pain and distress to the
animal.
2.0 Responsibility: It is the responsibility of the investigator, technician, or student
performing the task to follow the procedures established in this document.
3.0 Applicable Documents:
3.1 MSDS: Freund’s Complete Adjuvant
3.2 MSDS: Incomplete Fruend’s Adjuvant
3.3 SOP 03-003: Mouse Procedures.
3.4 SOP 02-005: Pain & Distress
3.5 SOP 01-011: Cell Line Disease Surveillance
3.6 SOP 03-013: Ascites Production in Mice
4.0 Special Considerations: The specific choice of the adjuvant is at the discretion of
the investigator but please be aware that the National Institutes of Health (N.I.H.)
has concluded that there is evidence that Freund’s Complete Adjuvant (FCA)
does cause distress and pain to the animal. Some negative effects of FCA are as
follows: granuloma formation, tissue necroses and sloughing, abscess formation
and fever. Also, FCA is considered a human biohazard and can cause painful
reactions if accidental inoculation or splashing should occur.
5.0 Other Considerations:
5.1 FCA should be used only in the initial immunization. Incomplete Freund’s
Adjuvant (IFA) should be used in future booster immunizations.
5.1.1 Other commercially produced adjuvants are available.
5.1.2 Dilution of adjuvant to antigen should be 1:1.
5.2 The material used as an antigen should be sterile.
5.2.1 When using cell lines, please refer to SOP 01-011: Cell Line Disease
Surveillance Policy and follow the procedures outlined in the
document.
5.3 When choosing an injection site be careful to chose an area that will not be
habitually traumatized by routine handling.
5.4 Volume injected into each inoculation site is dependent upon size of the
animal and site to be used:
SOP 03-014
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2)
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4)
5)
5.5
Location
Intramuscular (IM)
Subcutaneous (SQ)
Intradermal (ID)
Intraperitoneal (IP)
Footpad
Volume
0.05-0.1 ml/site
0.5 ml/site
0.02-0.05 ml/site
0.5 ml or less
0.025-0.05 ml/footpad
The number of inoculation sites used for SC or ID routes is dependent upon
species:
Species
# of sites
Mouse
2-4
Rabbit
10 or less
Rats
10 or less
Goat/Sheep
12 or less
6.0
Procedure:
6.1 Injection sites should be prepared using sterile technique. This is an important
step to help avoid formation of abscesses.
6.1.1 Clip the hair away from the injection site.
6.1.2 Clean injection site starting in the center and moving to the outer edge
using a sterile gauze pad and betadine solution. Repeat.
6.1.3 Re-wash injection site, from the center outward, using a sterile gauze
pad and alcohol.
6.2 If antigen is in an aqueous solution, emulsification with cold Freund’s can be
accomplished using two 3 cc syringes and a plastic 3 way stopcock. The 2
solutions can then be pushed back and forth between the syringes.
6.3 Immunizations should follow sterile technique SOP 03-003: Mouse
Procedures and fall within the guidelines stated above.
7.0
Post-Immunization Treatment:
7.1 All animals should be checked twice per day for any signs of pain and distress
as well as for any swelling or ulceration at the inoculation sites. SOP 02-005:
Pain & Distress.
7.2 Any signs of pain or distress should be brought to the attention of the animal
facility staff.
7.2.1 Animals displaying signs of pain and distress should be either
euthanized or treated with the following analgesics:
Species
Analgesic
Mouse
Butorphanol: 0.1-1.0 mg/kg, SQ/BID
Rabbits
Butorphanol: 0.1-0.5 mg/kg, SQ/BID
Rats
Butorphanol: 0.1-1.0 mg/kg, SQ/BID
Goat/Sheep
Butorphanol: 0.5 mg/kg, SQ/BID
SOP 03-014