Incomplete Freunds adjuvant

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 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. 

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. 

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)... 

Polyclonal Antibodies against FORL r purified polygalacturonase were raised in white rabbits. For the first immunization 200 jxg of purified protein in 300 jxl of distilled water was mixed with 200 1 of PBS and 500 n of complete Freund s adjuvcmt and injected intramusculary into the leg. Two subsequent intramuscular injections, each containing 300 fig of protein in 1 ml of incomplete Freund s adjuvant were given at 1 month intervals. Finally, the rabbit was bled 1 week later. The antisera, separated from blood by incubation at 37 "C, were stored in 1 ml fractions at -20 C. 

In contrast to certain oil emulsions (including Incomplete Freund s Adjuvant), the liposphere approach uses pharmaceutically acceptable biodegradable constituents. 

We have studied the effect of several adjuvants on the antibody responses to several of the peptides (52,53). In general, the highest responses were obtained in immunization of peptide with complete Freund s adjuvant. Immunizations without adjuvant (in PBS) or with Incomplete Freund s adjuvant usually gave little or no responses. 

Freund s adjuvant) used with an antigen to improve the immune response (antibody formation secondary to B-cell activation). Incomplete Freund s adjuvant does not contain the bacterial cells and is used to avoid an inflammatory response. See White, R.G., Factor affecting the antibody response, Br. Med. Bull. 19, 207-213, 1963 White, R.G., Antigen adjuvants, Mod. Trends Immunol. 2, 28-52, 1967 Myrvik, Q.N., Adjuvants, Ann. N.Y. Acad. Sci. 221, 324-330, 1974 Osebold, J.W., Mechanisms for action by immunologic adjuvants, J. Am. Vet. Med. Assoc. 181, 983-987, 1982 Warren, H.S., Vogel, F.R., and Chedid, L.A., Current status of... 

Various animal species can be used for antibody preparation rabbits are used most often, and goats for larger scale operations. For the first injection using complete Freund s adjuvant, intramuscular injection is recommended, whereas subsequent injections with incomplete Freund s adjuvant are carried out subcutaneously. 

Antigen Purified protein diluted to about 100 Xg/mL in PBS. Complete and incomplete Freund s adjuvant Two glass luer lock syringes 2 mL is the best size. 

The most popular and successful adjuvants have been the water in oil emulsions developed by Freund. The basic ingredients of light mineral oil (Bayol) and emulsifying agents mixtures such as Arlacel (A or C) are available commercially. The reagents are emulsified with either solutions or suspensions of the immunogen (incomplete Freund s adjuvant). The addition of mycobacteria (Mycobacterium butyricum, M. tuberculosis) in small amounts to the suspension (complete Freund s adjuvant) leads to a further enhancement of the immune response. This has been attributed to the increased local inflammatory response caused by the mycobacteria. ... 

Thtee C57BL/6 mice pet gtoup wete injected inttadermally with 50 pg of liposomal plasmid DNA (A, C) ot 50 pg free plasmid DNA (B, D) followed by a second treatment after 48 h. Three control mice received 50 pg of an antiviral peptide antigen (gp33) in incomplete Freund s adjuvant (IFA) inttadermally. Nine days after the first immunization, spleen cells were re-stimulated for 5 days and analyzed in a Cr-release assay on peptide labeled and unlabeled EL-4 cells. The spontaneous Cr-release was <14% (22). 

Indeed, lecithin-cholesterol liposomes successfully substituted for mineral-oil emulsion in the induction of delayed hypersensitivity In guinea pigs, administration of liposome-suspensions containing MDP and ovalbumin induced delayed skin reactivity similar to that observed following immunization in MDP-supplemented incomplete Freund s adjuvant (IFA) or in CFA.30,45 However, 6-0-acyl-MDP derivatives of appropriate fatty acid chain length (Cx2-C22> i 2, 3A), but not C2-C8 (9, 1, JJJ, showed in-... 

Kuroda et al. (2004) reported autoimmunity induced by adjuvant hydrocarbon oil components of some human and veterinary vaccines. This study showed that an injection of certain adjuvant hydrocarbon oils (pristane, incomplete Freund s adjuvant, or squa-lene) induced lupus-related autoantibodies to nuclear ribonucleo-proteins (nRNP)/Sm and Su in non-immune Balb/c mice. 

Assays to measure cell-mediated immunity (e.g., DTH) in macaques have produced inconsistent results. The antigens used in these studies and the concentrations of antigens used have varied between laboratories and the results have been highly variable. Most laboratories have used a cocktail of antigens that includes diphtheria, trichophyton, and Candida albicans in incomplete Freund s adjuvant (Price et al., 2004 and Chapter 3.1.3). 

Montanides Montanides are a group of oil/surfactant based adjuvants in which different surfactants are combined with a non-metaboKsable mineral oil, a metabolisable oil, or a mixture of the two. They are prepared for use as an emulsion with aqueous antigen solution. The surfactant for e.g. Montanide ISA 50 (ISA = Incomplete Seppic Adjuvant) is mannide oleate, a major component of the surfactant in Freund s adjuvants. The various Montanide ISA group of adjuvants are used as water-in-oil emulsions, oil-in-water emulsions, or water-in-oil-in-water emulsions. 

---