FREUND'S ADJUVANT, COMPLETE AND
INCOMPLETE
Product Number F 5881 AND F 5506
Storage Temperature 2-8 °C
Product Description
Appearance
F 5881 Clear amber liquid containing particulate matter
（dried cells）
F 5506 Clear amber liquid
Freund's Adjuvant is one of the most commonly used
adjuvants in research. It is used as a water-in-oil
emulsion. It is prepared from non-metabolizable oils
（paraffin oil and mannide monooleate）. If it also
contains killed Mycobacterium tuberculosis it is known
as Complete Freund's Adjuvant. Without the bacteria it
is Incomplete Freund's Adjuvant. First developed by
Jules Freund in the 1940's, Freund's Adjuvant is
designed to provide continuous release of antigens
necessary for stimulating a strong, persistent immune
response1,2,3 The main disadvantage of Freund's
Adjuvant is that it can cause granulomas, inflammation
at the inoculation site and lesions. The mycobacteria in
Complete Freund's attracts macrophages and other
cells to the injection site which enhances the immune
response. For this reason, the Complete Freund's
Adjuvant is used for the initial injections. To minimize
side-effects, Incomplete Freund's Adjuvant is used for
the boosts.
For comparisons of different adjuvant systems, see
references 4 and 5.
Reagents
Each ml of F 5881 contains 1 mg of heat-killed and
dried Mycobacterium tuberculosis （strain H37Ra, ATTC
25177）, 0.85 ml paraffin oil and 0.15 ml of mannide
monooleate.
Each ml of F 5506 contains 0.85 ml of paraffin oil and
0.15 ml of mannide monooleate.
Precautions and Disclaimer
Please consult the Material Safety Data Sheet for
handling recommendations before working with this
material.
Storage/Stability
Store in a cooler at 2-8 °C. Do not Freeze.
Procedure
1. If using Complete Freund’s Adjuvant, vortex or
shake to resuspend the Mycobacterium.
2. Mix antigens （preferably in saline） with an equal
volume of the adjuvant to form an emulsion. In
order to do this, vigorous and prolonged mixing is
needed. There are at least three methods which
can be used to accomplish this:
For small volumes the emulsion can be made in a
tube. Pipet the adjuvant in the tube first. Then,
while vortexing, add an equal volume of the antigen
solution. Vortex vigorously until a thick emulsion
forms.
For intermediate volumes, use two syringes
connected through a luer fitting. Ideally, a 3-way
valve should be used. Take the desired amount of
antigen solution into a glass syringe. The volume
should not fill more than half the syringe. Take an
equal volume of the adjuvant into another glass
syringe. Remove all air and connect the syringes
through the luer fitting to the 3-way valve. Adjust
the 3-way valve such that the connection is open
between the two syringes. Carefully depress the
plunger from the antigen solution first, pushing the
antigen into the oil of the adjuvant. Alternay push
the plungers, mixing the adjuvant and the antigen
solution into an emulsion. Continue until the
plungers are difficult to push.
For large volumes, use a tissue homogenizer to
make the emulsion. Add the adjuvant to the
homogenizer first. Run the homogenizer for a short
time to coat the inside with the adjuvant. Add an
equal volume of the antigen solution and run until a
thick emulsion forms.
3. The resulting emulsion should be very thick and a
drop of it should not disperse if tested by placing on
the surface of a saline solution.
4. Transfer the emulsion to a syringe （or remove one
syringe from the luer fitting if using the two-syringe
method）. Remove all the air. Add an appropriay
sized needle. The samples are now ready for
injection.6
References
1. Freund, J. and McDermott, K., Proc. Soc. Exp. Biol.
Med., 49, 548-553 （1942）
2. Freund, J., Ann. Rev. Microbiol., 1, 291 （1947）
3. Freund, J., Adv. Tuberc. Res., 7, 130 （1956）
4. Bennett, B. et al., J. Immuno. Meth., 153, 31-40
（1992）
5. Deeb, B.J. et al., J. Immuno. Meth., 152, 105-113
（1992）
6. Harlow, E. and Lane, D., Antibodies A Laboratory
Manual, （Cold Spring Harbor Laboratory, 1988）

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