Hapten-protein conjugates formation

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...

Figure 1 Principle of the competitive lA. In the first format with immobilized Ab (a) the plates are coated with Ab. Analyte and enzyme-labeled analyte compete for the Ab binding sites. In the second format a hapten-protein conjugate is immobilized in the soUd phase (b). This protein conjugate and the free analyte compete for the binding sites of the Ab in solution. (Reproduced from Dankwardt and Hock with permission from Food Technology and Biotechnology.)...

Synthesis of triazine haptens and hapten-protein conjugates. Simazine and atrazine were derivatized with mercaptopropionic add (mpa) at Rl, or aminohexanoic acid (aha) at R2, and these haptens were covalently linked to keyhole limpet hemoyanin (KLH), conalbumin (CON), or bovine serum albumin (BSA), by forming active esters with N-hydroxysucdnimide (g) (Figure 1, structures III and IV). This technique was also used to couple simazine-aminohexanoic add to calf intestine alkaline phosphatase (Figure 1, structure V), for use as the %aptenated enzyme in the EIA format described below. 

Fig. 1. Various possibilities of formation of hapten-protein conjugates in vivo...

In experimental studies on the formation of antibodies to a hapten-protein conjugate, it appears that the more immunogenic the protein, the better the response. Although proteins are usually the most efficient carriers, polypeptides, polysaccharides, lipid membranes, or even polynucleotides may be sufficient to impart im-munogenicity. In recent years, direct conjugation of simple chemicals to structural elements of cell membranes (especially of monocytes and macrophages) has been thought to play an important role in the induction of sensitization (de Weck 1975 Shearer 1975). The protein carrier or cell membrane structure of the host may contribute to the specificity of the response (partial autoimmunity). 

Carrier protein Macromolecule to which a hapten is conjugated, thereby enabling the hapten to stimulate the immune response. catELISA Similar to an ELISA, except that the assay detects catalysis as opposed to simple binding between hapten and antibody. The substrate for a reaction is bound to the surface of the microtitre plate, and putative catalytic antibodies are applied. Any product molecules formed are then detected by the addition of anti-product antibodies, usually in the form of a polyclonal mixture raised in rabbits. The ELISA is then completed in the usual way, with an anti-rabbit second antibody conjugated to an enzyme, and the formation of coloured product upon addition of the substrate for this enzyme. The intensity of this colour is then indicative of the amount of product formed, and thus catalytic antibodies are selected directly. 

We used 1,3-diketone hapten 1-carrier protein conjugate for immunization in our experiments to generate aldolase antibodies (Scheme 6.1) [4]. Class I aldolases use the e-amino group of a lysine in their active site to form a Schiff base vith one of their substrates and this substrate becomes the aldol donor substrate. Schiff-base formation reduces the activation energy for proton abstraction from the Ca atom and for subsequent enamine formation. The enamine, a carbon nucleophile, then reacts vith an aldehyde substrate, the aldol acceptor, to form a ne v C-C bond. The Schiff base is then hydrolyzed and the product is released [2]. The 1,3-diketone hapten acts as a mechanism-based trap of the requisite lysine residue in the active site and this lysine is necessary for formation of the essential enamine intermediate. The molecular steps involved in trapping the requisite lysine residue are essentially the same chemical steps as are involved in activating... 

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