Freunds Adjuvant

Freund s (1947, 1956) complete adjuvant is the most popular adjuvant. It consists of killed and dried mycobacterial cells suspended in an emulsifying oil. 

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Materials. Microspherical PGG glucan (Adjuvax, Alpha-Beta Technology, Worcester, MA) was prepared from Saccharomyces cereviseae strain R4 cells (11). Zymosan, cytochrome c (cyt c), bovine serum albumin (BSA), yeast alcohol dehydrogenase (ADH), Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA) were purchased from Sigma Chemical Co. (St. Louis, MO). 

The criterion for purity was a single band on a heavily loaded SDS-polyacrylamide electrophoresis gel. Antibodies were raised in rabbits in response to three intravenous injections each of 2mg. of laccase A with Freunds adjuvant, at 2 weekly intervals. Blood was removed 7d after the last injection and serum collected after coagulation of the blood cells. 

The route of antigen administration depends on the nature of the antigen itself, the animal species, the use of an adjuvant, and the immunological response. When producing antisera from rabbits, subcutaneous and intradermal administration are the most popular routes. Intramuscular injections can be used in the presence of Freund adjuvants because this route provides rapid access to the lymphatic system. The intravenous route is used with particulate antigens because injection can produce a response, which, although rapid, is not sustained. 

Both remacemide and its des-glycinyl-metabolite inhibit the inflammatory, mechanical hyperalgesia as well as the edema induced by injection of carrageenan or complete Freunds adjuvant into the rat hind paw (Asghar et al.,... 

There is also evidence that AMPA/kainate receptors may be involved in chronic pain following peripheral nerve injury AMPA receptor expression is upregulated and peaks 2 weeks after nerve ligation (Harris et al., 1996). Furthermore, GYKI52466 potently inhibits hyperalgesia in Freund adjuvant-induced chronic arthritis (Szekely et al., 1997). 

This test, performed in rats, is the classical model of inflammatory pain. Intraplantar injection of inflammatory stimuli such as carrageenan, kaolin, or complete Freund adjuvants (CFA) induces paw swelling and increased pain sensitivity. As pain stimulus pressure is applied on the inflammed paw and gradually increased until the animal responds by vocalisation or withdrawal of the paw. Analgesics increase the pressure threshold (Randall and Selitto, Arch. Int. Pharmacodyn. 1957, 111, 409-419). 

Figure 4. Binding of sH-lacto-Fl-tetraitol by rabbit serum (R31) in response to immunization with edestin-etNH-lacto-N-tetraose. Antigen in complete Freunds adjuvant was administered on Days 1, 38, and 58 (arrows). The binding assay is performed as described in the legend to Figure 2.

Raghavendra, V., Tanga, F. Y., and DeLeo, J. A. (2004). Complete Freunds adjuvant-induced peripheral inflammation evokes glial activation and proinflammatory cytokine expression in the... 

The first group underwent a conventional immunization procedure (6) in which one half mg of ECPs were administered subcutaneously in Complete Freunds Adjuvant (CFA) on day one and in Incomplete Freunds Adjuvant (ICFA) on day 7. The rabbits were boosted every 14 days for 160 days and serum collected 7 days after each boost. 

Week 2 Incomplete Freunds Adjuvant with RANTES protein was used as the immunogen (3), followed three days later by a boost of 10 pg of denatured RANTES protein (no adjuvant). 

Complete and Incomplete Freunds adjuvant were used in this procedure. These agents are extremely inflammatory. Always use protective eyewear and gloves when handling these agents. Several excellent alternative adjuvants that are far less stressful to the animals have been recently developed (3). The use of these new agents should be strongly considered. 

Mineral Salts Immunostimulatory adjuvants Lipid particles Particulate adjuvants Mucosal adjuvants Aluminium hydroxide, aluminium phosphate, calcium phosphate Saponins (e.g., QS21), MDP derivatives, bacterial DNA (CpG oligos), LPS, MPL and synthetic derivatives, lipopeptides, cytokines (e.g., GM-CSF, IL-2, IL-12) Liposomes, virosomes, iscoms, cochleates, emulsions (e.g., Freunds adjuvant, SAF, MF59 ) Poloxamer particles, virus-like particles, PLG microparticles Cholera toxin (CT), mutant toxin (e.g., LTK63, LTR72), heat labile enterotoxin (LT), microparticles, polymerized liposomes, chitosan... 

Production of Hybridoma Cell Lines. Eight-week-old female BALB/c mice were injected three times intraperitoneally with 500 xg of human serum albumin (HSA) at 2-week intervals. The first injection was in complete Freund adjuvant (1 1, v/v), and the succeeding two injections were given in incomplete Freund adjuvant (1 1, v/v). The mice were allowed to rest for 30-60 days and injected intravenously with 50 xg of HSA. Four days later, spleen cells from these mice were fused with SP2/0-Agl4 myeloma cells by using polyethylene glycol, as previously described (38). Hy-bridomas of interest were cloned by limiting dilution. 

To improve the specific response against microcystin immimoconjugates, synthetic lipopeptides were used as adjuvants and were found to invoke a greater immune response than the use of classical adjuvants such as Freunds adjuvant. However, an ELISA for the detection of free microcystin was not developed using these antibodies. Cross-reactivities of various microcystin variants and nodn-larin with monoclonal antibodies have been found affecting to the specificity of these antibodies for the recognition of the mentioned toxins. The number of purified microcystin variants that have been tested by ELISA using monoclonal antibodies shows marked differences between methods. 

Booster injections with the same amount of peptide conjugate emulsified in Incomplete Freunds Adjuvant is given every second or third week. Blood can be collected following the second booster injection and serum prepared by standard procedures Normally, high-titer antipeptide antisera are obtained after two booster injections... 

CpG-ODN 7909 in incomplete Freund adjuvant Melan-A pieptide Melanoma P-I (CP/GSK)... 

Three month old male New Zealand rabbits were immunized with irradiated B. jararaca venom. Prime injection was of Img in complete Freund adjuvant. The second was of the same venom amount in incomplete adjuvant and the booster consisted of Img toxoid in saline solution. 

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