Haptens, dinitrophenyl

Specifically, an appropriately designed 2,4-dinitrophenyl-hapten derivative of fluorescent traws-4,4 -diaminostilbene (DAS) was squeezed between two large globular proteins lysozyme (Lys) from one side and anti-2,4,6-trinitrophenyl antibody (anti-TNP) from the other side, in order to provide the desired constricted environment to restrict traws/ds-stilbene isomerization within the anti-TN P-DNP-DAS-Lys adduct (Figure 10.13). 

Tatsu et a/.(106) reported a novel immunosensor using immobilized liposomes doped with carboxyfluorescein and dinitrophenyl (DNP) hapten on the tip of an optical fiber. On complement-mediated immunolysis by anti-DNP-antibody, the fluorescent signal of the liberated carboxyfluorescein was measured. 

Antibodies Affinity labeling of antibody combining sites as illustrated by anti-dinitrophenyl antibodies, 46, 479 p-azoben-zenearsonate antibody, 46, 492 affinity cross-linking of heavy and light chains, 46, 501 bivalent affinity labeling haptens in the formation of model immune complexes, 46, 505 DNP-based diazoketones and azides, 46, 508 labeling of antilactose antibody, 46, 516. 

K. Balakrishnan, S. Q. Mehdi, and H. M. McConnell, Availability of dinitrophenylated lipid haptens for specific antibody binding depends on the physical properties of host bilayer membranes, J. Biol. 

An enzyme immunoassay using adenosine deaminase as the enzyme label has been described (318). Potentiometric rate measurements were made with an ammonia gas-sensing electrode. The immunoassay system employed is based on competition between a model haptenic group, dinitrophenyl (DNP) covalently coupled to adenosine deaminase, and free DNP hapten for the available binding sites on the anti-DNP antibody molecules. The detection limit is 50 ng antibody. 

A procedure introduced by us for steroid-protein conjugates was the estimation of the remaining free amino groups with the dinitrophenylation technique of Sanger. e-Dinitrophenyllysine was not isolated but was estimated directly by spectrophotometry after ether extraction of the acid hydrolyzate. A control with unsubstituted carrier was always run concomitantly, and the difference between the two was taken to be the extent of substitution by hapten. 

After blotting of the proteins, they are dinitrophenylated (Section 12.5). This hapten is then detected with anti-DNP antibodies (Miles, Inc.j by one of the methods discussed below. Detectability by this method is at least equal to that of autoradiography (< 1 ng). The intensity of the staining, however, is not representative of the stoichiometry of the protein in the blot. Kittler et al. (1984) haptenated the amino groups of the blotted proteins with pyridoxal-5 -phosphate. This method requires no organic solvents and can also be used with Zetabind membranes. 

Similarly, many different photoactivable haptens can be synthesized. Keller et al. (1989) prepared a 2,4-dinitrophenyl (DNP)-linker-aryl azide compound by a simple method from commercially... 

Polymer (2) was used as a test substance. It contains the dinitrophenyl-moiety, a classical hapten (DNP). Production of DNP-specific antibodies was measured by a radio-immunoassay (antigen-binding test). 

Schlossman SF, Levine H (1967) Immunochemical studies on delayed and arthus-type hypersensitivity reactions. I. The relationship between antigenic determinant size and antibody combining site size. J Immunol 98 211-219 Schlossman SF, Yaron A, Ben-Efraim S, Sober HA (1965) Immunogenicity of a series of, N-DNP-L-lysines. Biochemistry 4 1638-1645 Schlossmann SF, Levine H, Yarou A (1968) Studies on the specificity of antibody to 2,4-dinitrophenyl-poly-L-lysines. Biochemistry 7 1-7 Schneider CH, de Week AL (1967) The reaction of benzylpenicillin with carbohydrates at neutral pH with a note on the immunogenicity of hapten polysaccharide conjugates. Im-munochemistry 4 331-343... 

Figure 2.6 illustrates a typical curve for the binding of hapten by a purified rabbit antihapten antibody (73). The binding curves for a homogeneous Waldenstrom macroglobulin with specific activity toward the 2,4-dinitrophenyl (Dnp) group, and for some of the substructures of the macroglobulin, are shown in Fig. 2.7 (57). 

This method was first applied to antibodies by Velick et al. (56) and has since found widespread application, particularly in studies of antibodies directed to the dinitrophenyl and trinitrophenyl groups. Its major advantages are rapidity of measurement, which is essentially limited only by the rate of temperature equilibration, and a requirement for small amounts of antibody. (A typical concentration of antibody is 50 /ug/ml.) A disadvantage is that the method is essentially restricted to purified antibody. If, for example, an IgG fraction containing 10% of specific antibody is used, not more than 10% of the total fluorescence can be quenched by hapten. This would greatly decrease the accuracy of the measurements. 

Perhaps the most important and unique application of the method is in ascertaining when the nitroxide radical is actually present within the active site of the antibody. For example, one can synthesize haptens in which the nitroxide radical is spatially separated from the dinitrophenyl group by the insertion of chemical groups, e.g. (—CH2—) . If the radical is sufficiently far removed from the dinitrophenyl group it will not be located within the antibody combining site when the hapten is combined with antibody. Under these circumstances the paramagnetic resonance spectrum corresponds to that of the unbound free radical. 

Valentine and Green examined purified rabbit anti-2,4-dinitrophenyl (anti-DNP) antibodies of the IgG class, complexed with univalent hapten, by electron microscopy and could see only small globular... 

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