Antibodies to haptens

Figure 1 Schematic of the quasi-equihbria using heterologous haptens in coating antigen immunoassay formats. Ka represents the equilibrium constant for binding of antibody (Y) to target analyte (A). Kh is the equilibrium constant for the binding of antibody to hapten-protein conjugate (H-) immobihzed on a solid phase...

The antisera which were collected at ca. 100 days were first characterized for STXOL affinity (33). Equilibrium dialysis (34) gives a good measure of both antibody-to-hapten affinity ( gj useful range for RIA development 10 L/mole) and antibody... 

At the EM level, detection usually involves using a probe (oligonucleotide) in which a hapten has been incorporated. Incorporation of the hapten does not interfere with the hybridization of the complementary sequences. The next step is the binding of a reporter (may be an antibody) to the hapten. The reporter is then subjected to a binding molecule (may be a secondary antibody) that is coupled with an electron-dense material such as colloidal gold for visualization. Nonetheless, the many affinity-detection and immunodetection systems developed for immuno-cytochemistry may now with ingenuity be applied to molecular biology at the EM level. 

The library of natural catalysts has very little to offer for the catalysis of Diels-Alder (and the reverse) reactions (Diels-Alderases)[139]. For this reason one of the intriguing areas of biomimickry, namely the formation and use of antibodies exhibiting catalytic activity, has focused on [4 + 2] reactions to try to furnish proteins possessing useful catalytic properties. Thus in early studies a polyclonal catalytic antibody raised to hapten (57)[140] showed a modest rate enhancement for the reaction depicted in Scheme 48. 

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. 

Transition states of this tetrahedral nature have now been mimicked effectively by a range of stable analogues, including phosphonic acids, phosphonate esters, a-difluoroketones, and hydroxymethylene functional groups (Jacobs, 1991). Lerner s group elicited antibodies to a tetrahedral anionic phosphonate hapten [3] (Appendix entry 2.9)2 whilst Schultz s group isolated a protein with high affinity for p-nitrophenyl cholyl phosphate [4] (Fig. 4) (Appendix entry 3.2). 

The first example of bait and switch for catalytic antibodies was provided by Shokat (Shokat et al., 1989), whose antibody 43D4-3D12 raised to hapten [7] was able to catalyse the /3-elimination of [8] to give the trans-tnone [9] with a rate acceleration of 8.8 x 104 over background (Fig. 7 Appendix entry 8.1). 

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.

Fig. 25) (Baldwin, 1976). By raising antibodies to the charged hapten [67], Janda and co-workers produced an abzyme which accelerated 6-exo attack of the racemic epoxide to yield exclusively the disfavoured tetrahydropyran product [68] and in an enantiomerically pure form (Appendix entry 14.1) (Janda et al 1993). 

Li et al. (1997) have discussed the use of catalytic antibodies to control the reactivity of carbocations. At an entry level, the acyclic olefinic sulfonate ester [72] is converted into the cyclic alcohol [73] (98%) using antibody 4C6 raised to hapten [73] with only 2% of cyclohexene produced (Appendix entry 15.1) (Li et al, 1994). 

Fig. 29 Formation of isomeric decalins [71 ]—[73] by cyclization of a terpenoid alcohol catalysed by antibody HA5-19A4 raised to hapten [82]. The transition state [83a] has the leaving group in the equatorial position, as favoured by the Stork-Eschenmoser...

Table 4 Kinetic parameters for those antibodies raised against phosphonates [88-91] which effect the resolution of the fluorinated alcohols [84-87]. The configuration of the disastereoisomerically pure product from each antibody-catalysed process was shown to correspond to that of the antibody-inducing hapten.

More recently, Janda has described the production of a galactopyranosidase antibody in response to hapten [96]. This was designed to accommodate several features of the transition state for glycoside hydrolysis notably a flattened half-chair conformation and substantial sp2 character at the anomeric position. Some 100 clones were isolated in response to immunization with [96] and used to generate a cDNA library for display on the surface of phage (Appendix entry 7.3) (Janda et al., 1997). Rather than proceed to the normal screening for turnover, Janda then created a suicide substrate system to trap the catalytic species. 

Ross, A. H., Baltimore, D., and Eisen, H. N. (1981) Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to synthetic hapten. Nature 294, 654-656. 

The concept of catalytic antibodies was suggested succinctly by Jencks. If complementarity between the active site and the transition state contributes significantly to enzymatic catalysis, it should be possible to synthesize an enzyme by constructing such an active site. One way to do this is to prepare an antibody to a haptenic group which resembles the transition state of a given reaction. The combining sites of such antibodies should be complementary to the transition state and should cause an acceleration by forcing bound substrates to resemble the transition state. ... 

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