Incomplete Freund’s adjuvant

Freund s incomplete adjuvant (FIA) is a similar product. It differs from FCA in that it lacks the mycobacterial component and, consequently, displays somewhat lesser adjuvanticity. The mode of action of FIA is largely attributed to depot formation. The mycobacterial components in FCA have additional direct immunostimulatory activities. Although it is one of the most potent adjuvant substances known, FCA is too toxic for human use. 

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). 

Table 10.22. Some vaccine preparations in which Freund s incomplete adjuvant (FIA) was used as an adjuvant...

In C3H/HeN mice bearing MH134 hepatoma, monophosphoryl lipids A from P. gingivalis or S. minnesota Re 595 increased the survival of mice when administered in combination with tumor cell lysates and Freund s incomplete adjuvant [31],... 

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)... 

Figure 18.2 T-cell dependent antibody response in immature cynomolgus monkeys at various postnatal ages. Keyhole limpet hemocyanin (KLH) was administered intrader-mally at 100 jLLg in Freunds s incomplete adjuvant. Note that a clear antibody response is present at about 3 months of age followed by an increased response after second immunization at about 6 months of age. Data represent mean values + SEM of 5 to 6 animals (males and females combined).

S., Revisiting Freund s incomplete adjuvant for vaccines in the developing world. Trends Paristol. 21, 412 14, 2005. 

Repeat step 8-7 14 days later but inject only 0.3 to 0.5 ml of the emulsion. Prepare fresh emulsion each time that injections are given and make all emulsions after the first one using Freund s incomplete adjuvant in place of the complete adjuvant. 

Administer similar booster injections in Freund s incomplete adjuvant after 8 and 16 wk. 

Production of Polyclonal Anti-Plcloram Antibody. Picloram antisera was obtained from New Zealand White rabbits following the protocol described by Hall et al. (15). The rabbits were injected subcutaneously with an emulsion consisting of 0.5 to 1.0 mg immunogen dissolved in 0.5 mL of PBS and an equal volume of Freund s complete adjuvant. The injections were repeated 3, 6, and 10 days after the initial injection, substituting Freund s incomplete adjuvant for complete adjuvant. A booster injection was given one month after the initial injection and was repeated at monthly intervals thereafter. The rabbits were bled for antibody titer determinations 10 days after each boost. Antisera for picloram immunoassay development were prepared from a single bleed in each case. 

Nitro substituted carboxylic acids, N,N-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide were obtained from the Aldrich Chemical Co. (Milwaukee, WI). Avidin-labeled horseradish peroxidase, biotinylated anti-rabbit IgG, ovalbumin ( M.W. 45,000), bovine serum albumin (M.W. 66,000) Freund s complete adjuvant, Freund s incomplete adjuvant, and o-phenylenediamine were purchased from Sigma Chemical Co.(St. Louis, MO). All solvents were reagent grade. DME (1,2-dimethoxyethane) was dried by distillation from sodium-potassium amalgam/benzophenone ketyl. 

New Zealand white rabbits were injected subcutaneously at multiple sites with an emulsion consisting of 0.100 mg of BSA-hapten immunogen in 0.50 mL of phosphate buffer(PBS) and 0.50 mL of Freund s complete adjuvant. After 30 days a booster injection of 0.100 mg of immunogen in 0.50 mL of PBS buffer and 0.50 mL of Freund s incomplete adjuvant was given. Ten days after the boost the rabbits were bled and antibody titers were determined. In some cases additional boost were given after 10 day intervals to maintain high antibody titers. 

Female, New Zealand white rabbits were each immunized with an emulsion of the methoprene immunogen (200 fig per animal, 53 molecules of methoprene per molecule of protein) in 250 fib phosphate buffered saline and 250 fib Freund s complete adjuvant. A booster shot of the immunogen (200 fig per animal) in Freund s incomplete adjuvant was given to each animal one month after the initial immunization and again two weeks after the first booster shot. Rabbit anti-methoprene antiserum was collected and used to develop a competition enzyme-linked immunosorbent assay or cELISA for methoprene. 

Anit-Maduramicin Antibody Production. New Zealand white rabbits were hyperimmunized with maduramicin-BSA in Freund s complete adjuvant and boosted biweekly with Freund s incomplete adjuvant four weeks after the initial immunization. Weekly bleeds were performed. Serum titre was monitored by indirect enzyme immunoassay described below. The antibody was produced through a contract with Hazleton Laboratory (Vienna, Virginia). 

Development of Hybridomas that Secrete Anti-TPT Antibodies. Approximately 100-pg KLH-TPT was dissolved in PBS and emulsified with an equal volume of Freund s incomplete adjuvant (Sigma, St. Louis, MO). Female Balb/c mice (Harlan, Indianapolis, IN) were immunized with 200-pL emulsion by i.p. injection every 3 weeks. Animals were bled via the saphenous vein 7 days after each dose of KLH-TPT. Whole blood was collected and centrifuged. The resulting plasma was used to assess anti-TPT activity via a previously validated HPLC assay [10]. Briefly, plasma was diluted with PBS and incubated with 250-ng/mL TPT for 2h. Free TPT... 

Water-in-oil emulsions such as Freund s complete adjuvant, Freund s incomplete adjuvant. The complete adjuvant contains QdX- aDlQ6. Mycobacterium tuberculosis bacteria whereas the incomplete does not. Both have the basis of an immune modulator, e.g., branched glucose polymers or methylated bovine serum albumin (Pierce s AdjuPrimeTM). 

JIAO, x.-D., CHENG, s., HU, Y.-H. and SUN, L. (2010) Comparative study of the effects of aluminium adjuvants and Freund s incomplete adjuvant on the immune response to an Edwardsiella tarda major antigen. Vaccine 28,1832-1837. 

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