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Antibody Production
Adjuvants
Carrier Proteins
Antibody Production
Animals produce antibodies in response to proteins or other molecules (termed immunogens) recognized as foreign by their immune system. The antibodies are secreted into the plasma by B cells that are able to interact with the immunogen. The intensity of the response is determined by several factors including the size of the immunogen molecule, its chemical characteristics, and how different it is from the animal's own proteins. Most natural immunogens are proteins with a molecular weight above 5 kDa that come from sources phylogenically far removed from the host animal (i.e., human proteins injected into rabbits or goats). It is important to use very highly purified proteins as immunogens, since the animal will produce antibodies to even small amounts of impurities present as well as to the major component. The antibody response increases with repeated exposure to the immunogen, so a series of injections at regular intervals is needed to achieve both high levels of antibody production and antibodies of high affinity.
Even immunogenic molecules may not generate the level of antibody desired. To increase the intensity of the immune response immunogens are combined with complex mixtures called adjuvants. These mixtures contain agents that enhance the immune response without generating unwanted antibodies. If the immunogen is still unable to generate an acceptable response, it may be conjugated to a carrier protein that is more immunogenic.
Protocols for preparing immunogens, immunization of animals, and collection of antiserum may be found in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, NY, 1988) pp. 55-120 (Product CodeA 2926).
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Adjuvants
Adjuvants are mixtures of natural or synthetic compounds that, when administered with antigens, enhance the immune response. Adjuvants are used to (1) stimulate an immune response to an antigen that is not inherently immunogenic, (2) increase the intensity of the immune response, (3) preferentially stimulate either a cellular or a humoral response (i.e., protection from disease versus antibody production). Adjuvants have four main modes of action: enhanced antigen uptake and localization, extended antigen release, macrophage activation, and T and B cell stimulation. The most commonly used adjuvants fall into six categories: mineral salts, oil emulsions, microbacterial products, saponins, synthetic products and cytokines.
Table I gives examples of various adjuvants and their properties. Applications are given in
Table II.
A more extensive discussion of adjuvants and their use in immunization
protocols is given in Immunology Methods Manual, vol. 2, I. Lefkovits, ed.,
Academic Press, San Diego, CA, 1997, ch. 13. Immunology Methods Manual is
available as a four volume set, (Product Code
Z37,435-0); on CD-ROM, (Product Code
Z37,436-9); or both, (Product Code
Z37,437-7
).
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Carrier Proteins
Small molecules such as drugs, organic compounds, and peptides and oligosaccharides with a molecular weight of less than 2-5 kDa are not usually immunogenic, even when administered in the presence of adjuvant. In order to generate an immune response to these compounds, it is necessary to attach them to a protein or other compound, termed a carrier, that is immunogenic. When attached to a carrier protein the small molecule immunogen is called a hapten. Haptens are also conjugated to carrier proteins for use immunoassays. The carrier protein provides a means of attaching the hapten to a solid support such as a microtiter plate or nitrocellulose membrane. When attached to agarose they may be used for purification of the anti-hapten antibodies. They may also be used to create a multivalent antigen that will be able to form large antigen-antibody complexes. When choosing carrier proteins, remember that the animal will form antibodies to the carrier protein as well as to the attached hapten. It is therefore important to select a carrier protein for immunization that is unrelated to proteins that may be found in the assay sample. If haptens are being conjugated for both immunization and assay, the two carrier proteins should be as different as possible. This allows the antiserum to be used without having to isolate the anti-hapten antibodies from the anti-carrier antibodies.
KLH
Keyhole limpet hemocyanin (KLH) is a respiratory protein found in mollusks. Its large size makes it very immunogenic, and the large number of lysine residues available for conjugation make it very useful as a carrier for haptens. The phylogenic separation between mammals and mollusks increases the immunogenicity and reduces the risk of cross-reactivity between antibodies against the KLH carrier and naturally occurring proteins in mammalian samples.
KLH is offered both in its native form, for conjugation via amines, and succinylated, for conjugation via carboxyl groups. Succinylated KLH may be conjugated to a hapten containing amine groups (such as a peptide) via cross-linking with carbodiimide between the newly introduced carboxyl groups of KLH and the amine groups of the hapten.
M.W.: 8-9 x 106 Da
KLH is a polymeric protein that can undergo reversible dissociation into subunits. Because of this, the actual molecular weight is not generally used for calculation of the molar ratio of hapten to carrier. For these calculations, 1 mole of KLH is defined as 100,000 gm. Using this definition, the extinction coefficient of a 1 mg/ml (10 µM) solution at 280 nm is 2.02.
Protocols for conjugation of haptens to carrier proteins may be found in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, NY, 1988) pp. 78-87 (Product Code A 2926).
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