Recommendations before starting:
The inoculum should be free of extraneous microbial contamination; filtration of the antigen before mixing with the adjuvant is recommended.
Preparation of Aluminum hydroxide-immunogens mix is easier than other adjuvants as it does not require laborious emulsification.
1. Ensure complete re-suspension of Aluminum hydroxide adjuvant by vigorous shaking of the vial before use.
2. Dilute antigen mixture in saline buffer or phosphate buffer for a final immunogen concentration of 10-100 µg/100 µL.
3. Mix Aluminum hydroxide adjuvant with an equal volume of antigen solution for a 1:1 ratio:
a. Add Aluminum hydroxide dropwise with constant mixing to the immunogen solution.
b. Pipet up and down several times to ensure correct absorption of antigen by alum adjuvant and incubate 5-10 min.
NOTE: Ratio can be optimized from 1:1 (100 µL adjuvant per 100 µL antigen) to 1:9 (100 µL adjuvant per 900 µL antigen).
4. Inject into the animal according to table 1 below; the volume depends on the site of injection.
Typical routes of administration include subcutaneous (SC), intramuscular (IM), intradermal (ID) or intraperitoneal (IP)
| Species | Max vol. / Site | Primary Injection | Subsequent Injection(s) |
| Mice, hamsters | 0.1 mL | SC | SC |
| Mice, hamsters | 0.05 mL | IM∆ | IM∆ |
| Mice | 0.5 mL | IP× | SC, IM∆ |
| Guinea pigs, rats | 0.2 mL | SC, IM∆ | SC, IM∆ |
| Rabbits | 0.25 mL | SC, IM | SC, IM |
| Rabbits | 0.025 mL | ID SC, | IM |
Sheep, goats, donkeys,
pigs, chickens | 0.5 mL | SC, IM | |
∆ Not recommended in general, particularly for viscous adjuvants.
× Not recommended for pAb production.
Table 1: Maximum volumes for injection of immunogen/adjuvant
mixtures per site of injection for different animal species.
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