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The use of carrier proteins to boost the immune response

A hapten is a small molecule, typically with a molecular weight of less than 1-5kDa, which stimulates an immune response resulting in the production of antibodies only when it is attached to a larger carrier molecule. It is unable to generate an immune response on its own, since its size and lack of complexity preclude this.
Hapten-carrier protein complexes are typically used in antibody production programs for the generation of either polyclonal or monoclonal antibodies. Following immunization of an animal, usually a mouse or a rabbit, with the complex, B-cells produce antibodies against epitopes on both the hapten and the carrier protein.

  • Polyclonal antibodies are purified directly from the animal’s immune serum, which will contain a population of B-cells, each of which produces an antibody against a particular antigenic epitope. Only a fraction of the antibody population will specifically recognize the hapten, and therefore immunogen affinity purification may be required to enrich the antibody of interest. Polyclonal antibodies are subject to variability between animals and over time, and a permanent supply cannot be guaranteed.
  • Monoclonal antibodies are produced using hybridoma technology. B-cells are isolated and then fused with myeloma cells from the same host species, to give rise to immortalized cell lines which have an unlimited capacity to produce a specific antibody clone. Hybridoma clones can be grown in cell culture and antibodies harvested from the tissue culture supernatant, or they can be implanted into the peritoneal cavity of a mouse and antibodies isolated from the ascites fluid. These antibodies can then be purified and concentrated if necessary.

When selecting a suitable carrier protein, its immunogenicity and solubility must both be considered. The most commonly used carriers are Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA) and Ovalbumin.

Carrier protein Source Molecular weight (kDa)

Keyhole Limpet Hemocyanin

(KLH)

Keyhole Limpet

(Megathura crenulate)

Composed of multiple subunits

KLH1 ~360kDa

KLH2 ~390kDa

Bovine Serum Albumin (BSA)

Bovine

67kDa

Ovalbumin

Chicken egg white

45kDa

Formation of a hapten-carrier protein complex often necessitates chemical modification of the hapten, which can require specialist knowledge. To facilitate the quick and easy production of hapten conjugates, we have developed our imm-Link™ product range. To produce the conjugate, the hapten is simply pipetted into a vial of lyophilized mixture containing the carrier protein and all the necessary conjugation chemistry, and incubated. The conjugate is next dialyzed in the supplied dialysis cartridge to remove unwanted by products, and is then recovered and used. The design of the dialysis cartridge ensures that the hapten conjugate is recovered in high yield.

The imm-Link™ conjugation process

The imm-Link™ conjugation process. A solution of the hapten is pipetted into a vial of lyophilized mixture, which contains the carrier protein. After incubation and a simple dialysis, the hapten conjugate is ready to use.

Our imm-Link™ kits target a range of different functional groups and, with three different carrier proteins available, provide high levels of flexibility. The reagents are freeze-dried, and therefore ship at ambient temperature and have a long shelf-life. Furthermore, like all our products, the kits are rigorously QC tested to ensure consistent high quality and excellent batch-to-batch reproducibility.

Chemistry Source Molecular weight (kDa)

Carboxyl

BSA, Ovalbumin, KLH

Glutamic acid, aspartic acid or free carboxyl groups

Sulfhydryl

BSA, Ovalbumin, KLH

Cysteines or free sulfhydryl groups (SH)

Amine

BSA, Ovalbumin, KLH

Lysines or primary amines (NH2)

The imm-Link™ product range

Imm-Link™ kits for ELISA

Although the primary purpose of our imm-Link™ kits is to generate reagents which maximize an immune response, they can also be used to produce antigen-carrier protein conjugates which are ideally suited for an ELISA. If the antigen is small, and is to be used for plate-coating in an antigen-down ELISA, it must be bound to a larger carrier protein to allow adsorption to the microplate.

Once the microplate has been coated, it is washed to remove any unbound antigen, and is then blocked and washed again before the addition of an antigen-specific antibody. Indirect detection relies on the binding of a labeled, anti-species detection antibody, while direct detection avoids the need for secondary antibodies since the primary antibody is instead directly labeled with the detection moiety. Our Lightning-Link® product range enables direct antibody labeling via free amine groups, and includes kits for labeling antibodies with enzymes, biotin, streptavidin, fluorescent dyes or fluorescent proteins.

Antigen-down ELISA format, with direct detection

Antigen-down ELISA format, with direct detection. During an antigen-down ELISA, the microplate is coated with the antigen. If the antigen is small, it must be bound to a larger carrier protein to allow adsorption. Direct detection relies on the use of a labeled primary antibody.

For more information about our imm-Link™ kits, Lightning-Link® Antibody Labeling Kits, or any of our products, please contact us.