Hapten antibody specificity

Incidence of interferences observed in the radioimmunoassays are fairly insignificant by virtue of the highly specific hapten-antibody complexation reaction, and... 

Fig. 4. Classification of reported noncompetitive immunoassays for haptens based on the assay principle. (A) Assays that include a chemical modification of hapten to allow sandwich-type detection. (B1) Improved single-antibody immunometric assays that separate immune complex and excess labeled antibody, either by using a hapten-immobilized affinity column or based on differences in their physical properties. (B2) A variation of single-antibody immunometric assays based on masking of unoccupied antibody by an immunoreactive macromolecule followed by selective capture and detection of the hapten-occupied antibody. (C) Assays employing a probe molecule specific to a hapten-antibody complex.

We (K1) attempted to develop a noncompetitive assay based on the anti-idiotype antibodies for a conjugated bile acid metabolite, ursodeoxycholic acid 7-A-acetyl-glucosaminide (UDCA 7-NAG), which is expected to serve as a diagnostic index for an autoimmune disease, primary biliary cirrhosis. In our assay, the hapten UDCA 7-NAG, a /3-type antibody, and a biotin-labeled a-type antibody were simultaneously added to a microtiter plate coated with an F(ab )2 fragment of a specific anti-UDCA 7-NAG antibody, then incubated at room temperature for 8 h. Bound biotin was then detected with HRP-labeled streptavidin, whose enzyme activity was measured using o-phenylenediamine/H202 as a substrate. This noncompetitive assay system provided a subfemtomole-order sensitivity (detection limit 118 amol) that was 7 times lower than the competitive immunoassay using the same anti-hapten antibody (K2), even with a common colorimetric detection (Fig. 13). Somewhat improved specificity was also obtained namely, better... 

Development of such idiotype-dependent assays requires much labor and a long period because two-step antibody production is necessary first, immunization with a hapten-carrier conjugate to produce a specific anti-hapten antibody, then a second immunization with the anti-hapten antibody for generating two types of anti-idiotype antibodies. Furthermore, a suitable combination between a-type and /3-type antibodies, whose binding to the anti-hapten antibody is competitive, has to be found. 

Using an antibody specifically recognizing the antigen-antibody complex, more direct noncompetitive hapten immunoassays, which can be regarded as semi two-site immunometric assay, could be established (S3). Figure 14 depicts two typical procedures of noncompetitive assays using anti-metatype antibodies, which are based on principle C in Fig. 4. 

V4. Voss, Jr., E. W., Miklasz, S. D., Petrossian, A., and Dombrink-Kurzman, M. A., Polyclonal antibodies specific for liganded active site (metatype) of a high affinity anti-hapten monoclonal antibody. Mol. Immunol 25, 751-759 (1988). 

Antigens not only induce an immune response, but will also react with antibodies existing in a living organism. These two properties are different. Small molecules like pesticides are not able to induce the production of antibodies. They can only react with antibodies already present. They are called haptens. To generate antibodies specific to a hapten, haptens must be covalently bonded to a protein (e.g. BSA Bovine Serum Albumin) or polysaccharide. Therefore, the hapten must have reactive sites. On the other hand, this modification in the structure must not influence the specificity of the antibodies that will be produced. 

A major application of monoclonal antibodies is in clinical assays for drugs, bacterial and viral products, tumor antigens, hormones, and other circulating proteins. Their use in conjunction with immunoassays (Box 31-C) has provided increased specificity and sensitivity. Another major application is to observe binding of antibodies to specific proteins by electron microscopy. The location of specific receptor proteins can be established - as can the locations of ribosomal proteins and many other cellular components (Fig. 29-1). Monoclonal antibodies to acetylcholine receptors have been shown to induce symptoms of myasthenia gravis (Box 31-D), supporting the autoimmune origin of that disease. 1 Monoclonal antibodies specific for such a small hapten as mercuric ion have been isolated.k... 

The antibody titers seen in rabbits immunized with J5. typhimurium O-antigen-specific saccharide-BSA conjugates are shown in Table II. The characteristics of the antibody responses in rabbits and mice with respect to anti-hapten antibody response (16, 25, 60-63) can be summarized as follows ... 

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. 

A brilliant emitter. Certain naphthalene derivatives exhibit a weak yellow fluorescence when they are in a highly polar environment (such as water) and an intense blue fluorescence when they are in a markedly nonpolar environment (such as hexane). The binding of e-dansyl-lysine to specific antibody is accompanied by a marked increase in its fluorescence intensity and a shift in color from yellow to blue. What does this finding reveal about the hapten-antibody complex ... 

Much of Landsteiner s pioneer work was carried out with haptens that were aromatic amines. The compounds were converted to diazonium salts with nitrous acid and aUowed to react with proteins at alkaline pH (approximately 9). Reaction occurred primarily with histidine, tyrosine, and tryptophan residues of the protein carrier. For a representative procedure, see Kabat (p. 799 seq.). An interesting application of this procedure was the preparation of a chloramphenicol-protein conjugate which was used to elicit antibodies specific for chloramphenicol. In this case, a prior reduction of the nitro group of chloramphenicol to an amino group was required. As early as 1937, carcinogenic compounds were conjugated to protein carriers by means of their isocyanate derivatives which were prepared from amines. Immune sera were raised, and their properties were studied. - ... 

Haptens.—Haptens have been prepared from steroid hormones by reactions at C-3 or C-17, or in the pregnane side-chain. Antibody specificity is often improved, however, by anchoring the steroid through a middle-ring site to the protein, so that both ends of the steroid component of the complex are exposed for recognition in the antibody-forming process. 

Because of the multiplicity of metabolites that can be formed during some biotransformation reactions, it is not always possible to obtain a specific antibody to a hapten. Antibodies that recognize a family of related molecules (i.e., parent compounds and closely related metabolites or environmental degradation products) are useful to determine total immunologically reactive material for screening purposes. Such was the case for the antisera prepared against dieldrin by the... 

Several ELISA formats have been considered, and the one currently being tested is called the "hapten tracer" method. First introduced to this project by Dr. Freya Jung while a postdoctoral fellow in Dr. Hammock s laboratory, this method uses microtiter plates coated with a commercial preparation of goat anti-mouse antibody. Enzyme-labeled hapten competes with analyte for receptor sites on a mouse monoclonal antibody specific for the analyte. The amount of enzyme left after washing is inversely proportional to the... 

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