Detection of immune responses to haptens

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. 

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. 

The resulting epitope density may depend on the number of lysine groups in the particular protein, and this will in turn affect the immunogenicity of the antigen. Trifluoroacyl adducts have been detected on the outer surface of hepatocytes, presumably as a result of the hapten-complex processing and delivery by MHC I, which is described above. The fact that the production of the trifluoroacetyl chloride is part of the major metabolic pathway and that the majority of patients produce trifluoroacylated proteins suggests that it is differences in the immune surveillance system or immune responsiveness, which determine which patients will succumb to the immunotoxic effect. 

The highly specific and sensitive recognition of antigens by antibodies can be used for imunoaffinity purification. Antibodies, trapped within a silica gel, bind a specific molecule from a mixture of compounds. Small molecules (haptens) have to be bound to a macro-molecular carrier such as bovine serum albumin (BSA) to induce an immune response and stimulate the production of antibodies. These antibodies are then able to recognize specifically the target analyte (hapten) and have been used for the detection of various chemicals such as fluorescein, dinitrophenyl (DNP), dinitrobenzene (DNB), and even TNT (Wang, 1993 Aharonson, 1994 Jordan, 1996 Bronshtein, 2000 Lan, 2000 Vazquez-Lira, 2003). 

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