Modified haptens

As a rule, simple chemicals which are potent sensitizers are also highly reactive and can readily be shown to form conjugates with proteins in vivo. Those chemicals unable to form covalent bonds with proteins in vitro show no or only a very low incidence of sensitization. The extent to which a simple chemical is reversibly bound to serum proteins such as albumin has no apparent influence on its ability to sensitize. In several instances, the antibodies formed on sensitization with a simple chemical in vivo are specific not for the original compound injected, but for the modified haptenic structure formed on covalent binding to a protein carrier. 

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.. 

Raising the pi of macromolecules also can significantly alter the immune response toward them upon in vivo administration. Cationized proteins (those modified with diamines to increase their net charge or pi) are known to generate an increased immune response compared to their native forms (Muckerheide et al., 1987a, b Apple et al., 1988 Domen et al., 1987 Domen and Hermanson, 1992). The use of cationized BSA as a carrier protein for hapten conjugation can result in a dramatically higher antibody response toward a coupled hapten (Chapter 19). 

Figure 22.16 SMPB-activated liposomes may be modified with peptide hapten molecules containing cysteine thiol groups. The resultant immunogen may be used for immunization purposes to generate an antibody...

In contrast to a-methyldopa-induced hemolytic anemia, most drug-induced hemolytic anemia, especially for penicillins [34] and cephalosporins [35], involves drug-dependent antibodies, presumably because the drug acts as a hapten to directly modify erythrocytes or form immune complexes [36], However, there are many examples where a drug, such as nomifensine, induces both drug- (or metabolite)-dependent and drug-... 

The most interesting developments involve catalysis of simple aldol reactions. The key to reactive immunisation is the use of a hapten that is chemically reactive, rather than a passive template. This means that (i) relevant chemistry is going on during the course of antibody induction, which thus happens in the presence of intermediates involved in the reaction, and so may be modified to favor the formation of antibodies which bind these intermediates (and perhaps transition states leading to them). Furthermore (ii) it becomes possible to select for antibodies that react with, rather than just bind, to the hapten. The system used for the development of aldolase antibodies is outlined in Scheme 2... 

Autoantibodies to red blood cells and autoimmune hemolytic anemia have been observed in patients treated with numerous drugs, including procainamide, chlor-propaminde, captopril, cefalexin, penicillin, and methyldopa (Logue et al., 1970 Kleinman et al., 1984). Hydralazine- and procainamide-induced autoantibodies may also result in SLE. Approximately 20% of patients administered methyldopa for several weeks for the treatment of essential hypertension developed a dose-related titer and incidence of autoantibodies to erythrocytes, 1% of which presented with hemolytic anemia. Methlydopa does not appear to act as a hapten but appears to act by modifying erythrocyte surface antigens. IgG autoantibodies then develop against the modified erythrocytes. 

However, if primary antibodies of the same species belong to different IgG isotypes, they can be selectively detected with secondary antibodies directed against the corresponding isotype (see Sect. 8.2). If the primary antibodies are of the same species and of the same IgG isotype, they can be modified via their haptenylation. Haptenylated primary antibodies are subsequently visualized with the use of secondary antibodies recognizing the corresponding hapten (see Sect. 8.3). 

The bait and switch methodology deploys a hapten to act as a bait . This bait is a modified substrate that incorporates ionic functions intended to represent the coulombic distribution expected in the transition state. It is thereby designed to induce complementary, oppositely charged residues in the combining site of antibodies produced by the response of the immune system to this hapten. The catalytic ability of these antibodies is then sought by a subsequent switch to the real substrate and screening for product formation, as described above. 

The modified single-antibody immunometric assays discussed below are based on principle B2 in Fig. 4. In these assays, unoccupied antibody-binding sites are masked by reaction with a multiply hapten-labeled macromolecule to permit subsequent selective determination of hapten-occupied antibodies. 

Extracellular antigens are detected by APCs, such as lymphocytes, macrophages, and dendritic cells in interstitial fluid and blood. These detect the hapten and engulf the whole antigenic complex. Then, when inside the APC, the complex is partly dismantled and peptides attached to proteins similar to immunoglobulins, known as MHC II. The modified peptide-hapten complex is moved to the surface of the APC and presented as a complex with MHC to T-helper cells (CD4+), which activates and instructs the APC to make antibodies to the hapten and also B cells (memory cells with "memory" of the hapten) to proliferate. These events lead to types I to III responses. 

Intracellular antigens could be modified internal proteins, which are continuously removed by the cell, the structure altered and attached to MHC I. This takes place in the rough endoplasmic reticulum. The protein/pep tide-hapten fragments are then presented to the external surface of the cell membrane as a complex with the MHC I. Then cytotoxic T cells (CD8+) accept the protein/peptide and destroy the cell. This mechanism gives rise to a type IV response. 

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