Adjuvants play an important role in the antibody production by acting as immunopotentiators. They augment immune response via different mechanisms depending upon the adjuvant such as ‘depot’ effect, antigen presentation, antigen targeting, immune activation or modulation and cell-mediated response. The goal of adjuvant for antibody production is that high affinity, high titer and high avidity (for polyclonal) antibodies are raised. Both humoral and cell mediated response are necessary to achieve efficient antibody production. There are hundreds of preparations for adjuvants described in literature depending upon specific needs of an investigator. Nevertheless, most commonly and widely used adjuvants for antibody productions are few as listed below.
Freund’s Complete Adjuvant
Freund’s Complete Adjuvant (FCA)1 is used as water-in-oil emulsion with antigen. It is most commonly used for primary immunization to raise polyclonal and/or monoclonal antibodies for research purposes. FCA contains non-metabolizable oils like paraffin, surfactant mannide monooleate, and heat killed mycobacteria. The non-metabolizable oil along with water or buffer that contains antigen forms water-in-oil emulsion and the antigen is present in aqueous phase. There are several methods for emulsion preparation like vortexing, homogenization and forcing two liquids through double hub needle to allow mixing of aqueous liquid and oil. The resulting water-in-oil emulsion enhances immune response by ‘depot’ effect as it retains antigen for longer time at site of injection and release antigen slowly for the immune response. Furthermore, heat-killed mycobacteria attract macrophages which results in delayed hypersensitivity immune response and this may lead to granuloma formation and lesions. Inspite of several substitutes for FCA, it still is the most common adjuvant used for initial injections for raising antibodies. Its not used for subsequent injections as it forms granulomas and lesions. For secondary and booster injections Freund’s incomplete adjuvant is used. Adjuvants are not used for intravenous injections as this can lead to anaphylaxis.
Freund’s Incomplete Adjuvant
Freund’s Incomplete Adjuvant used as water-in- oil emulsion with antigen for secondary and booster injections to raise polyclonal and monoclonal antibodies. Freund’s Incomplete Adjuvant lacks the heat-killed mycobacteria present in Freund’s Complete Adjuvant and thus has less toxic effects like granuloma formation or lesions. Freund’s incomplete adjuvant is used along with Freund’s Complete Adjuvant to raise polyclonal and /or monoclonal antibodies. Freund’s Complete Adjuvant is used in primary immunization where as Freund’s Incomplete adjuvant is used in secondary and booster injections. Freund’s Incomplete Adjuvant can be used for primary immunization if the antigen is strongly immunogenic.
RIBI Adjuvant system
It has been known for long that microbes, mycobacteria and lipopolysachharides (LPS) enhance immune response non-specifically. This has led to invention of Ribi adjuvant system also known as RAS system. Ribi adjuvants are oil-in-water emulsions in which the antigen is mixed with small volumes of metabolizable oil and the oil droplets are then emulsified in saline solution containing Tween 80 as surfactant 2. This formulation as such is poor adjuvant but when combined with immunostimulators like LPS, mycobacterial cell wall skeleton (CWS), trehalose 6, 6’-dimycolate (TDM) etc, the immunogenicity is enhanced 3,4. There are three formulations of RAS available commercially for raising polyclonal and monoclonal antibodies. These formulations vary depending upon the different immunostimulant present. TDM emulsion recommended to be used with strong immunogen. Trehalose 6, 6’-dimycolate (TDM) is the lipid component of mycobacterial cord factor and it potentiates humoral and cell mediated response with strong immunogen or other immunostimulators. Monophosphoryl lipid A (MPL) + TDM emulsion is another formulation used to raise antibodies. Monophosphoryl lipid A is modified form of lipid A derived from bacterial lipopolysaccharide (LPS) and the modified form has less endotoxic effect where as the immunostimulatory activity is retained. The third RAS emulsion used for raising antibodies is MPL+TDM+ CWS (cell wall skeleton). The cell wall skeleton (CWS) is derived from mycobacterial cell wall extract. The loosely bound lipids, proteins and carbohydrates are removed from mycobacterial cell wall extract to get CWS. The main component of CWS is muramyl dipeptide which augments both humoral and cell-mediated immune response. In general RAS adjuvant system has been found to be less potent than FCA system but offers the advantage of being less toxic when compared to FCA.
TiterMax
Titermax adjuvants form microparticulate water-in-oil emulsion with patented block polymers (CRL 8941, CRL 8300), metabolizable oil squaline, small amounts of emulsifier sorbitan monooleate 80 and microparticulate silica. It’s been known that the lipids induce delayed type hypersensitivity response and this has led to discovery of non-ionic block co-polymers5.These co-polymers are a type of surfactant molecules composed of linear blocks or chains of hydrophobic polyoxypropylene (POP) and hydrophilic polyoxyethylene (POE) in different proportions. The basic biological activity of these block-copolymers it that they concentrate more antigen on their surface and this activity leads to antigen presentation and enhances cell- mediated immune response. These polymers have prominent adjuvant activity through antigen presentation, complement activation and macrophage activation. CRL 8941 or other block polymers are coated with silica particles which stabilizes the emulsion and this property of titermax has advantage over other adjuvants where large amounts of toxic emulsifying agents are used. Thus titermax act a potent adjuvant with less toxic effects. The literature points out that with titermax, antibody production at par or even more can be achieved when compared to Freund’s complete adjuvant with less toxic effects.
References
1. Freund, J. (1951). Am J Clin Pathol 21:645-656
2. Ribi, E. et al (1975). Cell Immunol 16:1-10
3. Altman, A., Dixon, F. J. (1989). Adv Vet Sci Comp Med 33:301-343
4. Azuma, I., et al (1974). J Natl Cancer Inst 52:95-100
5. Hunter, R. L., and Bennett, B. (1986) Scad J Immunol 23:287-300
