CFA (Complete Freund’s Adjuvant) is provided as a ready-to-use, pale yellow liquid. CFA is available in two quantities: 10 ml; 6 x 10 ml. Each 10 ml CFA vial contains 10 mg heat killed dried Mycobacterium tuberculosis (strain H37Ra), 1.5 ml Mannide Monooleate and 8.5 ml Paraffin oil. As CFA contains particulate matter, it may appear slightly hazy.
Storage and Stability
CFA is shipped at room temperature and should be stored at 4˚C.
CFA is stable for 2 years when properly stored. DO NOT FREEZE.
Complete Freund's Adjuvant (CFA) consists of heat-killed Mycobacterium tuberculosis in a water-in-oil emulsion. CFA contains trehalose 6,6’ dimycolate (TDM), which stimulates Mincle. Additionally, CFA has ligands for TLR2, TLR4, and TLR9. Injection of antigen in CFA induces a Th1-dominated response when compared to injection in Incomplete Freund's Adjuvant (IFA), which lacks mycobacterial components and induces a Th2-dominated response. An essential component of CFA-mediated response is an intense inflammatory reaction at the site of antigen deposition. For most applications, CFA is usually only necessary for the initial immunization, while IFA is the adjuvant of choice for subsequent immunizations.
Preparation of Antigen-CFA Emulsions
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.
1. Mix equal volumes of antigen and CFA using a 21 gauge needle and syringe. A white emulsion will form immediately.
2. Mix vigorously for several minutes to form an emulsion with maximum stability.
Note: A stable emulsion is essential to produce high titers of antibodies.
3. Vortex just before use.
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).