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Antigens are preferentially diluted in saline or phosphate buffers. The amount of protein or conjugated peptide used for the primary immunization can be adjusted depending upon availability and immunogenicity of the antigen. For example, mice can be injected subcutaneously (s.c.) with 1 to 10 μg of endotoxin-free ovalbumin.
Mix vigorously for several minutes to form an emulsion with maximum stability.
Note: A stable emulsion is essential to produce high titers of antibodies.
The volume of injection depends of the site of administration.
Note: To avoid anaphylaxis, do not use adjuvants for intravenous injection.
Recommended maximum volumes for injection of antigen/adjuvant mixtures per site of injection for laboratory animals. (Reference: Lindblad EB., 2000. Freund's Adjuvants. In: Vaccine adjuvants: Preparation Methods and Research Protocols. Humana Press.Totowa, NJ).

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