Hapten amino

Haptens with an amino group. Amine groups in haptens, carrier proteins or both can be modified for conjugation through homo- or heterobifunctional crosslinkers such as acid anhydrides (e.g., succinic anhydride), diacid chlorides (e.g.. 

Table 6 Conjugation of an amino-containing hapten to a protein using the glutaraldehyde method...

Ahlfors, S.R., Sterner, O. and Hansson, C., Reactivity of contact allergenic haptens to amino acid residues in a model carrier peptide, and characterization of formed peptide-hapten adducts, Skin Pharmacol. Appl. Skin Physiol., 16, 59, 2003. 

Here the hapten (Scheme 2) is a 13-diketone, which incorporates structural features of both reactants - ketone donor and aldehyde acceptor (see below, Scheme 3) - in the aldol reaction of interest. In favorable cases the hapten reacts with the primary amino-group of a lysine residue in the complementary-determining region of an antibody to form a Schiffbase 5, which readily tautomerises to the more stable vinylogous amide 6. 

It is believed that nickel penetrates the skin and acts as a hapten, complexing with selected peptide and/or amino-acid ligands to distort intercellular or cellular proteins, stimulating a type IV delayed (cell-mediated) hypersensitivity reaction [398]. Nickel water-soluble salts, like nickel chloride and nickel sulphate, are strong sensitizers [213, 215], The chloride induced in sweat is apparently an important factor in dissolving the metallic nickel, permitting the soluble nickel salts to act. 

Fig. 6 Binding site details for antibody 48G7 complexed with hapten p-nitrophenyl 4-carboxybutanephosphonate (Patten et al., 1996). N.B. Amino acid residues in antibodies are identified by their presence in the light (L) or heavy (H) chains with a number denoting their sequence position from the N-terminus of the chain.

A similar bait and switch approach has been exploited for acyl-transfer reactions (Janda et al., 1990b, 1991c). The design of hapten [10] incorporates both a transition state mimic and the cationic pyridinium moiety, designed to induce the presence of a potential general acid/base or nucleophilic amino acid residue in the combining site, able to assist in catalysis of the hydrolysis of substrate [11] (Appendix entry 2.6). 

Fig. 8 The original hapten [10] demonstrated the utility of the bait and switch strategy in the generation of antibodies to hydrolyse the ester substrate [11]. Three haptens, [12]-[14], were designed to examine further the effectiveness of point charges in amino acid induction. Both charged haptens, [12] and [13], produced antibodies that catalysed the hydrolysis of [11], whereas the neutral hapten, [14], generated antibodies which bound the substrate unproductively.

Hilvert s group used the same hapten [26] with a different spacer to generate an antibody catalyst which has very different thermodynamic parameters. It has a high entropy of activation but an enthalpy lower than that of the wild-type enzyme (Table 1, Antibody 1F7, Appendix entry 13.2a) (Hilvert et al., 1988 Hilvert and Nared, 1988). Wilson has determined an X-ray crystal structure for the Fab fragment of this antibody in a binary complex with its TSA (Haynes et al., 1994) which shows that amino acid residues in the active site of the antibody catalyst faithfully complement the components of the conformationally ordered transition state analogue (Fig. 11) while a trapped water molecule is probably responsible for the adverse entropy of activation. Thus it appears that antibodies have emulated enzymes in finding contrasting solutions to the same catalytic problem. 

The first success in sequence-specific peptide cleavage by an antibody was claimed by Iverson (Iverson and Lemer, 1989). He used hapten [43] containing an inert Cora(trien) complexed to the secondary amino acid of a... 

Fig. 6. Principle of a noncompetitive hapten immunoassay based on quantitative biotinylation of the target molecule (A) biotinylation of a hapten with reactive amino group(s) and subsequent (B) sandwich-type immunoassay procedure.

This assay procedure has been applied to various haptens having a primary amino group, including leukotriene C4 (VI), T4 (P2), substance P (P2), endothelin (P2), and AGII (G2) (for structures, see Fig. 5A and 5C). These noncompetitive assays were 10-300-fold more sensitive than corresponding competitive assays (G2). The minimal detectable concentrations described in these papers were as follows 2 pg/ml ( 0.3 fmol/assay) for leukotriene C4, 14 pg/ml ( 0.2 fmol/assay) for T4, 6 pg/ml ( 0.4 fmol/assay) for substance P, and 20 pg/ml ( 0.8 fmol/assay) for endothelin. 

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