Freund’s complete adjuvance

Two goats were immunized three times during the first 2 weeks with 1 mg of the antigen emulsified in 1 ml of Freund s complete adjuvant at several subcutaneous sites near regional lymph centers. Booster injections of 3 mg of antigen were administered at monthly intervals. The animals were bled 7 days after each boost. After several months of immunization, the titer and affinity of the antibody response was judged sufficient for use. 

Jongwon C, Keun H, Suk-Hwan K, Kyung-Tae L, Hyeong-Kyu L. Hee-Juhn P. Kalopanaxsaponin A from Kalopanax pictus, a potent antioxidant in the rheumatoidal rat treated with Freund s complete adjuvant reagent. J Ethnopharmacol 2002 79 113-118. 

The adjuvanticity of oil emulsions was first recognized in the early 1900s. However, the first such product to gain widespread attention was Freund s complete adjuvant (FCA), developed in 1937. This product essentially contained a mixture of paraffin (i.e. mineral) oil with dead mycobacteria, formulated to form a water-in-oil emulsion. Arlacel A (mannide mono-oleate) is usually added as an emulsifier. 

Ichikawa, H., Armstrong, R.B. and Harber, L.C. (1981). Photoallergic contact dermatitis in guinea pigs improved induction technique using Freund s complete adjuvant. J. Invest. Dermatol. 76 498-501. 

An emulsion of the hapten (i.e., conjugate) in normal saline is prepared by mixing with an equal volume of Freund s complete adjuvant. The prepared emulsion is injected subcutaneously into four different sites in New Zealand albino rabbits. Six weeks after the initial injection, all the animals are placed on a regimen of weekly booster shots. After a period of six months, antiserum from these animals are harvested and dilutions of 1 10,000 to 1 30,000 produced 50% binding or more and is employed in the RIA. 

The guinea pig maximization test (GPMT) is a preferred method for the detection of skin sensitizers. It belongs to the class of adjuvant-tests, where the substance will be applied in Freund s complete adjuvant (FCA). The test is based on the possible induction of an immune response of the skin during an induction period (at least 1 week). This pretreatment of the subject will eventually result in a hypersensitive reaction during a further exposure, the so-called challenging phase. 

Very often, vaccines are formulated with certain substances to enhance the immune response. These substances are called adjuvants (from the Latin adju-vare, which means to help ). The most common adjuvants for human use are aluminum hydroxide, aluminum phosphate, and calcium phosphate. Other adjuvants being used include bacteria and cholesterol. Mineral oil emulsions are normally the adjuvants used in animal studies. The adjuvant known as Freund s complete adjuvant consists of killed tubercle bacilli in water-inmineral oil emulsion, and Freund s incomplete adjuvant is a water-in-oil emulsion. Both these adjuvants are effective in stimulating an immune response, but they cause unacceptable side effects in humans (see Table 4.2). 

There are a number of models for detecting allergic contact dermatitis in guinea pigs. The maximisation test developed by Magnussun and Kligman is the most widely used and employs both an intradermal and topical sensitisation phase, together with the non-specific stimulation of the immune system by the intradermal injection of Freund s complete adjuvant. 

Table 10.21. Some toxic effects sometimes noted when Freund s complete adjuvant (FCA) is administered to experimental animals...

Prepare a stable 1 1 (v/v) emulsion of an eluted glycoprotein (20-250 pg in a buffer, preferably PBS) and an adjuvant If Freund s complete adjuvant is used for the first intradermal injection, this can be done by subjecting the mixture in an Eppendorf tube to a short ultrasonic treatment (usually three to four pulses for 10-20 s each time). During sonication, the tube should be placed on ice. 

Three weeks after the first injection, immunize the animal subcutaneously The emulsion is prepared m the same quantity and in the same way as before, but instead of Freund s complete adjuvant, use Freund s incomplete adjuvant (or an alternative adjuvant of the new generation of adjuvants)... 

Mix 2 mL of the RIP solution with 2 mL of Freund s complete adjuvant and emulsify, using a high-speed mixer. 

When rabbits were immunized with the glycoconjugates suspended in Freund s complete adjuvant antibodies with specificity for the haptenic disaccharides were produced, as estimated in passive hemagglutination and complement-mediated bactericidal tests, and enzyme-linked immunosorbent assay (ELISA) (12-15). 

New Zealand white rabbits (2-4 per immunogen) were injected (3-6 doses) at monthly intervals with the indicated saccharide-protein conjugate (10 yg/dose) or heat-killed j>. typhimurium SH4809 (1 x 10 bacteria/dose). The glycoconjugates were suspended 1 1 in Freund s complete adjuvant. 

---