Adjuvant incomplete

The mixture is injected once a week for 3 weeks, and then the animal is maintained for 3 weeks without additional injections. Approximately 25-40 ml of blood is removed from the animal s ear vein to test for antibodies. One week after the first bleed the rabbit is boosted with half the antigen amount used earlier along with incomplete adjuvant incomplete adjuvant does not contain the mycobacteria. Serum is again removed 2 weeks after this injection and tested for antibody response. The enzyme-linked immunosorbent assay is used to determine if the titer (or antibody concentration) of the serum is sufficiently high to establish antibody binding to the antigen. This 3-week cycle is repeated as long as necessary to obtain the antibodies desired. 

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. 

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). 

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. 

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

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). 

After 10 days, booster injections of the same amount of antigen in incomplete adjuvant were given every 2 weeks for a total of seven injections. Antisera to dust fractions (AD) and to bract (AB) were pooled and concentrated approximately tenfold by 40% saturation with ammonium sulfate at 4°c. The precipitated... 

Rabbit Immunization. Six rabbits were sub-cutaneously injected with. 2 mg of the immunogen in Freund s adjuvent on days 0, 12, 26, 40 and 59 of the immunization schedule (see Table I). Complete adjuvent was used for the first three immunizations followed by incomplete adjuvent for the final two immunizations. The rabbits were bled as needed using ear vein puncture. 

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. 

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],... 

Most popular adjuvants are the incomplete and the complete forms of Freund. The incomplete adjuvant is a water-in-oil emulsion by which the antigen is slowly released, providing a long period of contact between antigen and immune system. The complete adjuvant contains heat-killed Mycobacteria that causes a local injury and granuloma formation, thus providing a nonspecific stimulus. 

ARS2 dissolved in PBS was emulsified with an equal volume of Freund s complete or incomplete adjuvant. The emulsion was s.c. injected into male JW rabbits at an initial dose of 1.0 mg, followed by 0.5 mg injections every 2 weeks. Serum was collected 2 weeks after the last booster injection. 

Repeat these injections biweekly for a further 4 wk, but make the emulsion with incomplete adjuvant. 

Freund s complete and incomplete adjuvants. Alternatively, other adjuvants can be used in countries where the use of Freund s adjuvants is not allowed. 

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)... 

Most antigens require an adjuvant to increase their immunogenicity and a number of formulations can be used. Regulations on their use should be consulted prior to embarking on a course of immunizations. For many years Freund s complete and incomplete adjuvant were the formulation of choice for all immunization work. In recent years welfare issues have been raised over the use of these adjuvants and a number of alternatives based on water-soluble bacterial cell wall components have become available. 

Freund s complete and incomplete adjuvants FCA is a suspension of heat-killed Mycobacterium tuberculosis in oil. This should be shaken well before use. FIA is the oil without the bacteria. Freund s adjuvants should be mixed with the aqueous immunogen solution/ suspension at a ratio between 1 2 and 2 1 and mixed until an emulsion is formed. This is most easily achieved by passage back and forth between two glass syringes connected by a three-way luer fitting. After a short time (1-5 min) the mixture should become noticeably stiffer and may then be administered. 

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. 

The word optimize is purposely used here. Herbicides have a certain level of innate activity. Adjuvants do not increase this activity but merely aid in the movement of an herbicide to an area or region of a plant where the herbicide will do the most good. It has clearly been established that surfactants do increase the apparent activity of herbicides (2, 4, 5, 10). The cause of this increase, however, is incompletely understood. Indeed, there may not be a simple explanation of the observed phenomenon. The answer may actually be a complex interaction of the various components. 

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. 

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).

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