Hapten immunogenicity

New Zealand white rabbits were injected subcutaneously at multiple sites with an emulsion consisting of 0.100 mg of BSA-hapten immunogen in 0.50 mL of phosphate buffer(PBS) and 0.50 mL of Freund s complete adjuvant. After 30 days a booster injection of 0.100 mg of immunogen in 0.50 mL of PBS buffer and 0.50 mL of Freund s incomplete adjuvant was given. Ten days after the boost the rabbits were bled and antibody titers were determined. In some cases additional boost were given after 10 day intervals to maintain high antibody titers. 

As a low molecular weight compound DAS is not significantly immunogenic, ie, it is a hapten and thus requires conjugation to a suitable antigenic carrier in order to eUcit a successfiil antibody response in animals. DAS treated with succinic anhydride results in the D AS—hemisuccinate (DAS—HMS) shown. 

Davio et al. (43) report efforts to obtain monoclonal antibodies (mAbs) to STX. Because STX is a small molecule of approximately 300 daltons, well below the size necessary for immunogenicity, a carrier molecule must be conjugated to the hapten (STX). This technique must minimize alterations of the antigenic form. For the anti-STX antibodies tested to date, the ratios of immunoassay response factor to pharmacological potency for various STX derivatives differ substantially, the immunoassay being virtually unresponsive to some of the common natural derivatives (44). 

Table 1 Guidelines for the design and synthesis of an immunogen hapten...

Figure 8 Structure of immunogen haptens for pyrethroids with spacer arm attachment at the a-position of the alcohol moiety. Since the whole pyrethroid molecule is available for recognition by the antibody, assays resulting from these immunogens were selective for the parent pyrethroids...

However, if a class-selective assay is desirable (for multi-analyte assays), the handle should be located at or near a position that differentiates members of the class and exposes features common to the class. Using the pyrethroid example, an ideal immunogen should retain the phenoxybenzyl moiety and link the protein from the distal acid end (Figure 9). Using such an immunogen hapten, a class-specific immunoassay was developed that was highly cross-reactive with the type I pyrethroids permethrin, phenothrin, resmethrin and bioresmethrin. ... 

Figure 9 Structure of the immunogen hapten used to generate antibodies for a type I pyrethroid class-selective assay. Pyrethroids lacking an a-cyano group are generally termed type I. This hapten exposed the features most common to type I pyrethroids, the phenoxybenzyl group, the cyclopropyl group and the lack of a cyano group, resulting in antibodies that recognized permethrin, phenothrin, resmethrin and bioresmethrin, but not cypermethrin...

Figure 11 Structure of the phenoxybenzoic acid (PBA) immunogen hapten. Conjugation to the protein through the aldehyde resulted in an immunogen that generated antibodies selective and sensitive for PBA...

Figure 13 Structures of haptens used for immunizing and coating antigens in a monoclonal antibody-based immunoassay for diuron. A sensitive assay was developed using coating hapten I that had the handle in a position different from the immunogen hapten. When the oxygen in the urea moiety of hapten I was replaced with a sulfur (hapten 11), increasing the heterology, even greater sensitivity was achieved...

Carrier Molecule Hapten Hapten-carrier Immunogen... 

Figure 19.1 Immunogens are made by the crosslinking of a hapten molecule with a carrier using a conjugation reagent.

Synthetic haptens mimicking some critical epitopic structures on larger macromolecules are often conjugated to carriers to create an immune response to the larger parent molecule. For instance, short peptide segments can be synthesized from the known sequence of a viral coat protein and coupled to a carrier to induce immunogenicity toward the native virus. This type of synthetic approach to immunogen production has become the basis of much of the current research into the creation of vaccines. 

Some synthetic carriers actually are designed to have low immunogenicity on their own to minimize the potential for antibody production against them. When a hapten is coupled to these molecules, the immune response is directed principally toward the modification, not at the carrier. This design approach guides most of the immune response toward the desired target and minimizes the production of carrier-specific antibodies. 

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