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IACUC / LARC STANDARD PROCEDURES
IMMUNIZATIONS TO DETERMINE IMMUNE STATUS AND ANTIBODY
ISOTYPE PRODUCTION IN MICE
(Posted November 2006)
Description of procedure:
Mice are injected intraperitoneally or subcutaneously once with protein
or peptide antigens in adjuvant. Note: foot pad injections are never done.
Typically, the adjuvant will be alum (aluminum potassium sulfate) and the
antigen will be Trinitrophenol (TNP)-conjugated to keyhole limpet hemocyanin
(KLH), although other protein antigens may be used (such as the recombinant
Leishmania LACK antigen, pigeon cytochrome C, or ovalbumin) and other adjuvants
may also be used (e.g.complete Freund's adjuvant - CFA). Mice are injected
i.p. or s.c. with the antigen/adjuvant precipitate (1-100 ug protein in
5-10% alum or other suitable adjuvant in 100-200 ul sterile phosphate buffered
saline) – when given subcutaneously, no more than 0.05mls will be
injected per site. The purpose of these experiments will generally be to
monitor the antibodies made by the mice in response to the foreign antigens.
25-50 ul of blood will be taken from each mouse (retro-orbital or tail vein)
at several time points. All blood collection will be performed under isoflurane
anesthesia. This is necessary to establish the increase in IgM antibody
titer and IgG antibody titer, both of which will be evaluated at several
time points. In some experiments, the mice are re-immunized after 4-6 weeks
and then bled several days after the booster immunization in order to monitor
secondary or memory antibody responses. This re-immunization will be performed
using the same antigen preparation as was used initially, unless the primary
adjuvant was complete Freund’s adjuvant, in which case the secondary
immunization will be performed with incomplete Freund’s adjuvant or
without adjuvant. The mice are typically euthanized at the end of the 14-day
period if only the primary immune response is being measured, although in
some instances they may be kept alive for a period of 2-6 months to study
long-term immune memory, after which they will then be euthanized per the
UCSF Guidelines on Euthanasia for Rodent Species.
Mice injected subcutaneously with complete Freund’s adjuvant will
be examined for ulceration at the injection site. Mice will be evaluated
for general appearance, ability to move normally around cage and reach food
and water, ability to eat and drink, grooming habits, skin color, lethargy,
aggression (animals will be housed individually if aggressive behavior is
shown). Mice will be evaluated by the Body Condition Scoring index (BCS).
Mice injected with complete Freund’s adjuvant will be examined 24
hrs and 3 days post-injection to monitor for ulceration at the injection
site. Immunized mice will be examined weekly thereafter.
No adverse reactions are anticipated. Mice injected with complete Freund’s
adjuvant may show skin irritation or less frequently ulceration at the injection
site. Although severe reactions are unusual, mice with ulceration at the
injection site or with a Body Condition score <2 will be euthanized.
Literature search words required:
Literature search was performed for refinement of this
Standard Procedure on Sept 1, 2006:
| Key Words |
Search Site |
Years Covered |
| Blood collection alternatives in
rodents |
Pubmed |
1966-2006 |
Agents:
- This procedure requires complete Freund’s adjuvant
and isoflurane anesthesia for blood collection. [specify whether anesthetic & analgesics
are required].
- All agents administered to animals should be listed
in the “Agents” section of RIO.
Adverse Effects:
Adverse
Effects |
| Procedure,
Agent or Phenotype |
Potential
Adverse Effects |
Management |
| CFA |
Subcutaneous: Skin irritation or
less frequently ulceration at injection site |
euthanasia |
| Retro-orbital blood collection |
Ocular infection or mechanical
trauma to the eye |
Consult with Veterinary Services
|
Monitoring Parameters |
| Monitoring Parameters |
Frequency |
PI/Lab will Document |
| General appearance, grooming habits & responsiveness |
Post-CFA mice monitored @24 hrs,
3 days post-injection and weekly thereafter for 2-8 months |
No |
| Ability to ambulate normally, eating
and drinking & BCS |
Post-CFA mice monitored @24 hrs,
3 days post-injection and weekly thereafter |
Only if animal evaluation of BCS
is instituted for any animal(s) |
| Mucous membrane color |
Post-CFA mice monitored @24 hrs,
3 days post-injection |
No |
| Describe
the conditions, complications, and criteria (e.g. uncontrolled
infection, loss of more than 15% body weight, etc.)
that would lead to removal of an animal from the study,
and describe how this will be accomplished (e.g. stopping
treatment, euthanasia). |
| Ulceration at injection site, BCS <2
, or irreparable trauma (as determined by the Veterinary
Service group) to eye during blood collection are criteria
for euthanasia. |
| For all investigators
housing animals with tumor formation, skin lesions, neurological
deficits, or that are in Category E, list the expected
characteristics/clinical presentations and endpoints of
the animal model and the criteria for euthanasia. Note:
The IACUC also requires such lists to be posted in the
respective animal rooms and monitored by the IACUC compliance
staff and LARC, to assure PI adherence to the endpoints
listed. |
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