| Name | Freund 's Adjuvant Incomplete |
| Synonyms | FICA F5506 abs9271 Freund's Adjuvant Freund 's Adjuvant Incomplete |
| Storage Condition | 2-8°C |
Freund's immune adjuvant
Immune adjuvants refer to certain substances, which can enhance the body's specific immune response to the antigen before or when injected with the antigen at the same time, that is, to assist the antigen and enhance the antigenicity, thereby obtaining a higher titer The role of antibodies. Immune adjuvant (immunogenic adjuvant) is one of the hot spots in vaccine research. Aluminum hydroxide, as the only human adjuvant, has been successfully used in vaccines for viral diseases such as hepatitis B virus. Immune adjuvant is a non-specific immune enhancer. It can enhance the body's immune response to antigen together with antigen or pre-injected into the body.
Freund's immune adjuvant is currently a commonly used type of immune adjuvant, which is divided into incomplete adjuvant and complete adjuvant. Freund's incomplete adjuvant is to mix an aqueous antigen solution with an oil (paraffin oil or peanut oil) in the same amount, plus an emulsifier (lanolin or Tween 80) to make a water-in-oil emulsion, which can increase the level of antibodies, And the duration is long. Freund's complete adjuvant is the addition of dead BCG vaccine to Freund's incomplete adjuvant, which can stimulate the proliferation of mononuclear macrophage system, increase antibody production and antigen uptake. Dead BCG in Freund's complete adjuvant stimulates T cells to elicit an immune response. The adjuvant was invented by Hungarian-American bacteriologist JulesThomas Freund(1891-1960), so it is named after it. Because the adjuvant contains mycobacteria, it is also called mycobacterial adjuvant. Those who do not contain mycobacteria are Freund's incomplete adjuvant.
mechanism of action
Immune adjuvant refers to a class of substances that can enhance antigenicity and immune response after being mixed with antigens and injected into animals. Effect of immune adjuvant:
① can enhance antigenicity of weak antigenic substances;
② Increase the level of specific circulating antibodies, or produce effective protective immunity;
③ Change the type of circulating antibody produced;
④ Enhance cell-mediated hypersensitivity;
⑤ Protect antigens (especially DNA and RNA) from decomposition by enzymes in the body.
Adjuvants can especially enhance their specific response to certain substances with low molecular weight polysaccharides or peptides with weak antigenicity.
The difference between Freund's incomplete adjuvant and Freund's complete adjuvant
Freund's complete adjuvant is a water-in-oil emulsion that can effectively induce the production of high titers of antibodies. Freund's complete adjuvant contains cell wall components of Mycobacterium tuberculosis, which can enhance the antibody response to antigens. Adjuvant activity comes from the sustained release of immunogens in oil droplets and stimulates the local immune response. Freund's incomplete adjuvant is used for primary immune stimulation. In order to reduce side effects, Freund's incomplete adjuvant without Mycobacterium tuberculosis components is used to strengthen immunity.
Freund's adjuvant preparation method
Freund's adjuvant is the most commonly used adjuvant in animal experiments, divided into incomplete Freund's adjuvant and complete Freund's adjuvant. Incomplete Freund's adjuvant is a mixture of liquid paraffin and lanolin. The component ratio is 1~5:1, which can be determined according to needs, usually 2:1. Incomplete adjuvant plus BCG (final concentration of 2~20mg/ml) or dead Mycobacterium tuberculosis, namely complete Freund's adjuvant (FCA). Generally, 1/2 volume of FCA plus 1/2 volume of antigen is used for emulsification during the first injection, and incomplete adjuvant or no adjuvant is used for the second or third injection. If no adjuvant is added, the amount of antigen will be increased by 10-20 times.
preparation method: place lanolin and paraffin oil in a container according to the proportion, mix them evenly with ultrasonic wave, sterilize them under high pressure, and store them at 4 ℃ for later use.
Before immunizing animals, first mix Freund's adjuvant and antigen in a certain proportion, and the volume ratio of adjuvant and antigen is generally 1:1 to prepare a "water-in-oil" emulsion. Because it is easy to emulsify SDS into water-in-oil antigen emulsion complex, it must be kept emulsified when injected into animals. The amount of antigen used varies according to the molecular weight of the antigen, immunogenicity and immune animals, and there is no uniform standard and fixed mode. Generally, 1mg of antigen is injected for the first time per rabbit (about 2kg weight) or per sheep (about 20kg weight), and then the amount of antigen is increased step by step, up to 3mg each time.
The emulsification of adjuvant and antigen can be carried out as follows:
(1) grinding method: first heat the adjuvant and put an appropriate amount into a sterile glass mortar. after cooling, slowly drip the same volume of antigen solution, and grind in the same direction while dropping, and the speed of dropping antigen should be slow. After all the antigens are added, continue to grind for a period of time to make it a milky white viscous water-in-oil emulsion. This method is suitable for the preparation of a large number of adjuvant antigens, the disadvantage is that the mortar wall adheres to a large number of emulsions, antigen loss is large.
(2) syringe mixing method: suck the same amount of Freund's adjuvant and antigen solution into the two syringes respectively, connect the two syringes with a thin hose, pay attention to drain the air, and then alternately push the syringe until a viscous emulsion is formed. The advantages of this method are easy to do aseptic operation, less antigen loss, suitable for the preparation of a small amount of antigen emulsion. But at the same time, it is difficult to emulsify completely. Individual antigens cannot be pushed at all with plastic syringes, and there is leakage with glass syringes. The prepared emulsifier can only be applicable after identification. The identification method is to drop the emulsifier into the cold water. If it is kept intact and not dispersed, it floats on the water surface in a droplet shape, that is, the emulsification is complete, and it is a qualified water-in-oil.
(3) ultrasound: if the laboratory conditions are better, for example, if there is an ultrasonic crusher, the ultrasonic frequency and time must be controlled. ultrasound is easy to excite some free radicals to resist the original unknown damage. The emulsification method depends on the antigen and the need.
main applications
1. Freund's adjuvant can be used to prepare water-in-oil emulsion of immunogen. Due to the slow release of antigen, the antigen in water-in-oil emulsion can stimulate a highly effective long-term antibody response.
2. Mycobacteria in Freund's complete adjuvant can attract macrophages and other cells to the injection site, thereby enhancing the immune response. For this reason, Freund's complete adjuvant was used for the initial injection and Freund's incomplete adjuvant for the subsequent booster injection.
3. Sigma's Freund's complete adjuvant specification: product number: F5506; Each ml contains 0.85mL of paraffin oil and 0.15mL of mannitol monoleic acid.