Antibodies hapten-specific

Key words Affinity chromatography, Affinity matrices, Antigen-specific antibodies, Hapten-specific antibodies, Specie-specific antibodies, Cross-reacting immunoglobulin, Sepharose, Affigel, IgM, IgA, Mannan binding protein, Jacalin, Protein A, Protein G, Protein L... 

Affinity chromatography has been used to isolate antigen-specific antibodies, hapten-specific antibodies, species-specific antibodies or to separate cross-reacting immunoglobulins from the antibody of interest. It has also been used to remove specific contaminants, e.g., serine proteases such as trypsin, thrombin, factor Xa or other contaminating proteins such as albumin. 

The concept of immunoassay was first described in 1945 when Landsteiner suggested that antibodies could bind selectively to small molecules (haptens) when they were conjugated to a larger carrier molecule. This hapten-specific concept was explored by Yalow and Berson in the late 1950s, and resulted in an immunoassay that was applied to insulin monitoring in humans. This pioneering work set the stage for the rapid advancement of immunochemical methods for clinical use. 

In an immune response, antibodies are produced and secreted by the B-lymphocytes in conjunction with the T, cells. In the majority of hapten-carrier systems, the B cells end up producing antibodies that are specific for both the hapten and the carrier. In these cases, the T lymphocytes will have specific-binding domains on the carrier, but will not recognize the hapten alone. In a kind of synergism, the B- and T-cells cooperate to induce a hapten-specific antibody response. After such an immune response has taken place, if the host is subsequently challenged with only the hapten, usually it will respond by producing hapten-specific antibodies from memory cells formed after the initial immunization. For a review of immunobiology (see Janeway, 2004). 

Another approach to this persistent problem relies on haptenylation of primary antibodies. Hapten (e.g., biotin, digoxigenin or any fluorophore) can be covalently bound to the antibody via A-hydroxysuccinimide esters (NHS-ES) (see Sect. 2.1), or conjugated employing monovalent IgG Fc-specific Fab fragments (see Sect. 2.2). Haptenylated primary antibodies can be subsequently visualized with the use of secondary antibodies recognizing the corresponding hapten (Fig. 8.5). Fluorophore-labeled primary antibodies can be directly visualized in a fluorescent microscope. 

Neuberger, M.S., Williams, G.T., Mitchell, E.B., et al. (1985). A hapten-specific chimeric immunoglobulin E antibody which exhibits human physiological effector function. 

Using 2-methylbenzimidazolecarbamate as a hapten, monoclonal antibodies that specifically bind fenbendazole, fenbendazole sulfone, oxfendazole, albendazole sulfone, and albendazole sulfoxide have been also produced (76). On the... 

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... 

When rabbits were immunized with the glycoconjugates suspended in Freund s complete adjuvant antibodies with specificity for the haptenic disaccharides were produced, as estimated in passive hemagglutination and complement-mediated bactericidal tests, and enzyme-linked immunosorbent assay (ELISA) (12-15). 

Fig. 10. Different techniques for competitive immuno-polymerase chain reaction of small-molecule compounds. (A) Using biotinylated haptens and a DNA-streptavidin nanocircle conjugate [105], hapten-DNA conjugates were synthesized and used for a competitive assay in a sample containing free hapten and capture antibody-coated surfaces [92]. (B) Hapten-coated microplates were simultaneously incubated with a sample containing free hapten and a hapten-specific antibody. Following competitive coupling, the immobilized antibody was subsequently detected by a species-specific antibody-DNA conjugate [93, 94].

Quantitative precipitation tests indicate both how much hapten-specific antibody is present and how much unconjugated protein would be required to absorb out all the antibodies directed against the protein alone. This absorption can then be done by incubating equivalence concentrations of antigen and serum and removing the precipitate. Absorption can also be done with protein-agarose affinity columns. In addition, the antibody can be purified with hapten-bearing affinity columns, from which hapten-specific antibody can be eluted with 2 M acetic acid or with excess hapten. 

Under certain circumstances, it may be advantageous to have both soluble and insoluble conjugates containing the same determinant group. The latter can be used for the isolation and purification of hapten-specific antibody. For a review of insoluble hapten-carrier conjugates, we refer the reader to Jakoby and Wilchek and Williams and Chase. ... 

Once sensitivity has been established, that is, once hapten-specific IgE-producing B cells have been formed, exposure to even small amounts of hapten can induce a cascade of events that lead to immediate reactions, such as anaphylaxis (210). Briefly, preformed IgE antibodies to drug determinants recognize the hapten-carrier complex and fix to the surface of mast cells or basophils, triggering the release of a series of mediators, such as histamine, neutral proteases, biologically active arachidonic acid products, and cytokines. This ultimately leads to a clinical spectrum that ranges from a mUd local reaction to anaphylactic shock. 

Fig. 2. Principle of ELISA. Antibodies elicited against a hapten-KLH conjugate are tested against a hapten-BSA conjugate. Only hapten-specific antibodies are detected. A blocking agent is used to prevent direct binding of antibodies to the solid support...

Purified nucleic acids are poor immunogens but can serve as high molecular weight haptens. Synthetic RNA DNA hybrids, such as poly(A) poly(dT), can serve as immunogens (Stollar and Rashtchian, 1987) and the antiserum obtained can be absorbed with ds RNA or denatured DNA. The remaining antibodies may specifically capture hybrids from solution or in competitive ELISA test. 

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