Hapten-carrier complexes

The interaction of a chemical (hapten) with epidermal proteins (carrier) can result in a hapten-carrier complex capable of activating skin-associated lymphoid tissue (sensitisation) and dissemination of antigen-specific T l)unphocytes (induction). Subsequent encoimter with the same or cross-reactive chemicals can result in the elicitation of a characteristic inflammatory skin reaction. The clinical condition is referred to as allergic contact dermatitis and is characterised by erythema, oedema, vesiculation and pruritus. Allergic contact sensitisation is, therefore, classed as a cell-mediated immunological response to chemicals that contact and penetrate the skin. 

Since keyhole limpets are marine creatures existing in a high-salt environment, KLH maintains its best stability and solubility in buffers containing at least 0.9 M NaCl (not 0.9%). As the concentration of NaCl is decreased below about 0.6 M, the protein begins to precipitate and denature. Conjugation reactions using KLH, therefore, should be done under high-salt conditions to preserve the solubility of the hapten—carrier complex. 

Once sensitivity has been established, that is, once hapten-specific IgE-producing B cells have been formed, exposure to even small amounts of hapten can induce a cascade of events that lead to immediate reactions, such as anaphylaxis (210). Briefly, preformed IgE antibodies to drug determinants recognize the hapten-carrier complex and fix to the surface of mast cells or basophils, triggering the release of a series of mediators, such as histamine, neutral proteases, biologically active arachidonic acid products, and cytokines. This ultimately leads to a clinical spectrum that ranges from a mUd local reaction to anaphylactic shock. 

In vivo, drug-protein (hapten-carrier) complexes can be taken up by APCs, are transported into local draining lymph nodes, and are processed and/or presented to naive T cells by MHCs on APCs. T cells with the appropriate specificity recognize these complexes and are induced to proliferate and expand as primed T cells. In addition, hapten-carrier complexes can be antigenic for B cells. Such hapten-carrier complex-specific B cells, in the presence of T cell help, proliferate and differentiate into plasma cells that produce different antibody isotypes. 

Macromolecules are haptenated for the preparation of (i) immunogenic hapten-carrier complexes for the production of anti-hapten antibodies (ii) reagents for the detection of haptens by EIA and, (iii) reagents which, in conjunction with anti-hapten antibodies, restrict the specificity or increase the applicability of solid-phase EIA or EIH. The properties required of the hapten-conjugates are quite different for the three different situations. The haptenation procedures will be described according to the functional group of the hapten. 

Closely linked to bioactivation is the formation of hapten-carrier complexes. Generally the binding capacity of reactive compounds to model carrier peptides or nucleophilic amino acids is determined. For this also a number of assays are available for instance using HPLC or LC, in combination with MS or NMR (Singh et al., 2004 Ahlfors et al., 2005). If the new chemical can be radiolabeled, binding to peptides can be easily detected. In cases where specific antibodies to the compound or to structurally related compounds are available, immunochemistry techniques such as Western blotting or even immuno-histology can be applied. 

Detection of immune responses to haptens is the same as that described above, but there are some special techniques now being used. A hapten-carrier complex can be prepared in vitro by combination in solution with a large protein such as albumin or a smaller molecule such as glutathione. These can then be used to coat a solid substrate. Alternatively, the protein carrier can be coated onto a substrate, the hapten added, and then the assay performed. This probably increases the amount of hapten that is available for antibody binding and minimizes that which is lost in the tertiary folding of the protein. 

The work outlined above, and the interpretation of the chemical findings, indicates that as far as any resultant determinants are concerned, aminolysis results in structures in which only the R1 side chain, the attached amide, and remnants of the p-lactam ring remain from the original cephalosporin molecule. The resultant penaldate- and any penamaldate-like structures finked to carrier protein therefore represent hapten-carrier complexes that may interact with side chain (Rl)-specific IgE antibodies in allergic responses to therapeutic dosage of cephalosporin drugs. 

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