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Protocol

Preparing and Using Adjuvants

Edward A. Greenfield

Nonspecific stimulators of the immune response are known as adjuvants. The judicious use of adju-
vants is essential to induce a strong antibody response to soluble antigens. The most commonly used
adjuvant for research work is Freund’s adjuvant. Described here is the preparation and use of Freund’s
adjuvant as well as four alternative adjuvants: Ribi, Hunter’s TiterMax, Magic Mouse, and
aluminum hydroxide.

MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental
Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at
http://cshprotocols.cshlp.org/site/recipes.

Reagents
Adjuvant of choice
Alum adjuvant (e.g., ADJU-PHOS; Sergeant Adjuvants)
Freund’s Complete adjuvant
Freund’s Incomplete adjuvant
Hunter’s TiterMax Gold (0.5-mL vial)
Store at 4˚C.
Magic Mouse Adjuvant (Creative Diagnostics)
Magic Mouse adjuvant is supplied as a ready-to-use solution and is shipped at ambient temperature. On
arrival, it should be stored at 2˚C–8˚C. The adjuvant is stable for up to 1 yr at 2˚C–8˚C.
Ribi Adjuvant System
Store at 4˚C–8˚C.
When beginning an immunization, choosing the correct adjuvant can be difficult. Generally, Freund’s adjuvant
should be used when small amounts of the immunogen are available. If large amounts are available or if the
compound is known to be highly immunogenic, then other adjuvants can be used. If the target site (epitope) is
conformational or discontinuous, the immunogen could require a hydrophilic environment to retain the
conformation of the targeted site. Water-based adjuvants would be a better choice for these types of antigens.
If any single adjuvant has been tried and the immune response in the animal has been weak, try switching to a
different class of adjuvant; an adjuvant that works well for one immunogen might not be the best choice for a
different immunogen. Additional information concerning adjuvant selection can be found in Stills (2005).

Antigen(s) of interest

From the Antibodies collection, edited by Edward A. Greenfield.

© 2019 Cold Spring Harbor Laboratory Press
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E.A. Greenfield

Bacillus pertussis, heat-killed (for Alum adjuvants only [optional; see Step 30])
Mice or other animals to be immunized
NaOH (0.25 N) (for Alum adjuvants only)
Phosphate-buffered saline (PBS) <R> (for Ribi, Hunter’s TiterMax, and Magic Mouse adjuvants only)
Saline (0.9%) (for Alum adjuvants only)
Equipment
Centrifuge
Needles, 18, 22, and/or 23 gauge
Syringes, glass, 3 cc
Syringes, plastic, 1 cc
Three-way stopcock (optional; see Steps 2.iv–viii)
Tissue homogenizer (optional; see Steps 2.ix–xi)
Tubes, conical, 50 mL
Vortexer

METHOD

Preparing Freund’s Adjuvant

The most commonly used adjuvant for research work is Freund’s adjuvant, a water-in-oil immunopotentiator com-
posed of mineral oil with the surfactant mannide monoleate. It is available in two forms: Complete (CFA) and
Incomplete (IFA) (Freund and McDermott 1942; Freund 1956). CFA contains heat-inactivated mycobacteria
(usually Mycobacterium tuberculosis), whereas IFA lacks the mycobacterial components. Fruend’s adjuvant attracts
macrophages and stimulates both cell-mediated and humoral immunity. To avoid the majority of the side effects, the
primary injection should be given in CFA, but all boosts should be performed in IFA. Because Freund’s adjuvants are
potentially harmful to humans, care should be taken during preparation and injection. The Freund’s/antigen emulsion
should be kept sterile to avoid Pasteurella bacterial infections, which can induce carbuncles at the injection site that
can rupture and compromise the health of the animal.

1. If using Complete Freund’s adjuvant, resuspend the M. tuberculosis by vortexing or shaking.

2. Using one of the following methods, mix protein antigens (preferably in saline) with an equal
volume of the adjuvant oil until an emulsion is formed.
To generate this emulsion, vigorous and prolonged mixing is needed.

For Small Volumes (<200 µL)

i. Add the adjuvant to the tube.

ii. While vortexing, add an equal volume of the antigen solution.
iii. Vortex vigorously until a thick emulsion develops.

For Intermediate Volumes (0.2–3 mL)

iv. Use two syringes connected through a Luer fitting (Fig. 1). A three-way valve is appropriate.
v. Using glass syringes, take equal volumes of the aqueous antigen and the adjuvant into two
different syringes.
Do not fill over one-half of the syringe capacity.

vi. Remove all air. Connect the syringes through the Luer fitting.
vii. Depress the plunger from the aqueous solution first, driving the antigen into the oil of
the adjuvant.
viii. Alternately push the plungers, mixing the adjuvant and the immunogen solution into an
emulsion. Continue until the syringe plungers are difficult to push.

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Preparing and Using Adjuvants

FIGURE 1. Preparing Freund’s adjuvant for injection. (A) Preparation of emulsion by passing from one syringe to
another. (B) Close-up of a well-mixed emulsion.

For Large Volumes (>3 mL)

ix. Add the adjuvant to a tissue homogenizer first.

x. Run the homogenizer for a short period to coat the inside with the adjuvant.
xi. Add the aqueous solution and run until a thick emulsion develops.
3. Test that the emulsion is properly made. It should be very thick and not disperse when a drop of it
is placed on the surface of a saline solution.
4. Transfer the emulsion to a syringe (or remove one of the syringes from the Luer fitting). Remove
all the air from the syringe. Add an appropriately sized needle.
The samples are now ready for injection. Freund’s (or any oil-based adjuvant) must never be given
intravenously.

Using Ribi Adjuvant

Ribi adjuvants are oil-in-water emulsions composed of a metabolizable oil (squalene) that is emulsified with saline
containing Tween 80. Like Freund’s adjuvant, Ribi also comes in two forms. One form contains mycobacterial
products plus bacterial monophosphoryl lipid A, whereas the second form lacks the mycobacterial products. Ribi
induces cytokine production by immune cells, leading to antigen uptake and presentation.

5. Warm the vial of Ribi adjuvant for 5–10 min at 40˚C–45˚C.

6. Add 2 mL of PBS directly into the Ribi vial through the rubber stopper.
7. Vortex the vial for 3 min at room temperature.
8. Aliquot 1 mL into individual tubes. Store at 4˚C until needed.
9. When ready to immunize animals, take one 1-mL aliquot. Mix 1:1 with an equal volume of
antigen dissolved in PBS.
10. Transfer the antigen/adjuvant emulsion to a 3-cc syringe with a 23-gauge needle. Remove
the bubbles.
11. Restrain or anesthetize the animal, and inject the antigen/adjuvant emulsion.
12. Administer booster immunizations at 4, 8, and 12 wk.
13. Bleed animals 10–14 d after last boost.

Using Hunter’s TiterMax Adjuvant

Hunter’s TiterMax is another oil/surfactant-based adjuvant that is water-in-oil based like Freund’s adjuvant. Unlike
Freund’s, it uses squalene and a synthetic, nonionic surfactant (copolymers of polyoxyethylene and polyoxypro-
pylene) with good protein-binding capacity. The surfactant is able to activate complement and bind complement
components that help target the antigen to follicular dendritic cells in the lymph nodes and spleen. TiterMax Gold

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E.A. Greenfield

is the latest incarnation, which works effectively in mice and rabbits. Depending on the antigen, it may work as well as
or better than Freund’s. The main benefit of TiterMax is that it uses copolymer-coated microparticles to form stable
emulsions with less oil. The resulting emulsion is less viscous than Freund’s adjuvant, making it easy to inject through
small needles, reducing the amount of inflammation at the injection site, and reducing the frequency of booster
injections.

14. Emulsify 0.5 mL of antigen dissolved in PBS directly with the Hunter’s TiterMax Gold adjuvant in
the 0.5-mL adjuvant vial. Mix.
This will be enough to immunize 20 mice.

15. Aliquot unused antigen/adjuvant emulsion. Store at 4˚C, 20˚C, or −80˚C until needed.
Reemulsify before using.

16. Transfer the antigen/adjuvant emulsion to a 1-mL plastic syringe. Remove any air bubbles.
17. Restrain or anesthetize the animal. Inject the antigen/adjuvant emulsion.
18. Administer two to three booster immunizations every 3–4 wk.
19. Bleed the animal 10–14 d after the final booster immunization to check serum titer.

Using Magic Mouse Adjuvant

Magic Mouse adjuvant is an aqueous suspension that has been tailor-made to induce the rapid production of high
titers of antibodies in mice. The adjuvant contains immune-stimulatory CpG DNAs (i.e., short oligonucleotides
containing unmethylated cytosine–guanine dinucleotides within a certain base context) that can be found natu-
rally in bacterial DNA. CpG activates the innate immune system through Toll-like Receptor 9 (TLR9) found on
macrophages. TLR9 triggering activates a signaling cascade leading to the production of proinflammatory cyto-
kines and activation of the innate immune system. Because different CpG DNA sequences activate the immune
systems of different animal species, Magic Mouse adjuvant is specifically designed for immunization of mice.
When mixed with an antigen, CpG DNA produces high titers of antigen-specific antibodies. Unlike water-in-oil
emulsions, Magic Mouse adjuvant preparation does not require sonication, heating, lyophilization, or homogeni-
zation, allowing the native conformation of the immunogen to be maintained. However, because Magic Mouse is
aqueous based, there is no depot effect, and frequent booster injections could be required. Magic Mouse is
nontoxic with no adverse effects. This can be useful for generating antibodies against conformational epitopes on
native antigens.

20. Calculate the total amount of immunogen required. Dilute the immunogen with PBS (or another
animal-compatible buffer) to twice what its final concentration will be in the immunogen–
adjuvant mixture.
The recommended immunogen dosage is 1–50 μg per injection for weak immunogens such as recombinant
homologous proteins and conjugated peptides (usually 5–10 μg per injection) and 0.1–10 μg per injection
for highly immunogenic immunogens such as inactivated viruses or recombinant viral proteins (usually 1–2
μg per injection).
21. Mix Magic Mouse adjuvant in its vial by gentle vortexing. Add the adjuvant to the immunogen at
a 1:1 ratio (v/v). Mix by gently pipetting up and down five times.
Immunogen–adjuvant mixes should be freshly prepared before injection and used immediately. It is normal
to see precipitation in the adjuvant or immunogen/adjuvant mixture. Mix well before drawing into a syringe
and inject as quickly as possible after drawing into the syringe. The total volume of immunogen–adjuvant
mix used per mouse is 50–100 µL.
22. Inject 100 µL of the immunogen–adjuvant mix into a quadriceps muscle of each mouse.
Subcutaneous or intradermal injections are also compatible with Magic Mouse adjuvant and will yield the
same results as intramuscular injection.
23. Boost animals 3 wk after the first immunization, following Steps 20–22.
The serum titer peaks around 35 d after the priming immunization.

24. Bleed from tail tips 2 wk after the first boost. Measure the antibody titers by ELISA.
ELISA titers should be in the range of 1:10,000–1:10,000,000.
See Troubleshooting.

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Preparing and Using Adjuvants

Preparing Aluminum Hydroxide (Alum) Adjuvant

Aluminum salts are good immunopotentiators in vaccines and can be used as adjuvants for generating an antibody
response. Aluminum hydroxide adjuvant, aluminum phosphate adjuvant, and alum are the most common aluminum
compounds used as adjuvants. For aluminum hydroxide, the immunogen is either adsorbed to preformed precipitants
or is trapped during precipitation. This protocol was adapted, with permission, from Chase (1967).

25. Prepare a 10% potassium alum solution in distilled H2O.

For long-term storage, filter-sterilize the solution and dispense in convenient volumes.

26. In a 50-mL conical tube, add 10 mL of 10% potassium alum. While vortexing, add 22.8 mL of
0.25 N NaOH dropwise. Incubate for 10 min at room temperature.
27. Centrifuge at 1000g for 10 min.
28. Remove and discard the supernatant. Add 50 mL of distilled H2O to the pellet. Resuspend the
aluminum hydroxide, Al(OH)3. Centrifuge at 1000g for 10 min.
29. Combine the Al(OH)3 adjuvant with the antigen. Dilute with 0.9% saline if necessary.
If the antigen is abundant, set up a titration of different amounts of the Al(OH)3 versus a constant amount of
antigen. One milligram of Al(OH)3 will bind
50–200 μg of protein antigen.
30. Optionally, induce a nonspecific stimulation of the immune response in this system by adding
heat-killed B. pertussis to the adjuvant–antigen solution. Use
4 × 109 organisms per 100 µL of
material to be injected.
31. Incubate the antigen–adjuvant solution for 20 min at room temperature. Centrifuge at 10,000g
for 10 min. Test the supernatant for the presence of the antigen to be certain that it has bound.
The samples are now ready for injection.

TROUBLESHOOTING

Problem (Step 24): The antibody titer is lower than required.

Solution: Boost the animals again on Day 42 after priming. Bleed again from the tail tips, and measure
the titer using ELISA.

RECIPE

Phosphate-Buffered Saline (PBS)

Final Final
Amount to add concentration Amount to add concentration
Reagent (for 1× solution) (1×) (for 10× stock) (10×)
NaCl 8g 137 mM 80 g 1.37 M
KCl 0.2 g 2.7 mM 2g 27 mM
Na2HPO4 1.44 g 10 mM 14.4 g 100 mM
KH2PO4 0.24 g 1.8 mM 2.4 g 18 mM
If necessary, PBS may be supplemented with the following:
CaCl2•2H2O 0.133 g 1 mM 1.33 g 10 mM
MgCl2•6H2O 0.10 g 0.5 mM 1.0 g 5 mM
PBS can be made as a 1× solution or as a 10× stock. To prepare 1 L of either 1× or 10× PBS, dissolve the
reagents listed above in 800 mL of H2O. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then
add H2O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at
15 psi (1.05 kg/cm2) on liquid cycle or by filter sterilization. Store PBS at room temperature.

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E.A. Greenfield

REFERENCES

Chase MW. 1967. Production of antiserum. In Methods in immunology and
immunochemistry (ed. Williams AC, Chase MW), Vol. 1, pp. 197–209.
Academic Press, London.
Freund J. 1956. The mode of action of immunologic adjuvants. Bibl Tuberc
7: 130–148.
Freund J, McDermott K. 1942. Sensitization to horse serum by means of
adjuvants. Proc Soc Exp Biol Med 49: 548–553.
Stills HF Jr. 2005. Adjuvants and antibody production: Dispelling the myths
associated with Freund’s complete and other adjuvants. ILAR J 46:
280–293.

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Preparing and Using Adjuvants

Edward A. Greenfield

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