Antibodies anti-hapten

Selected entries from Methods in Enzymology [vol, page(s)] Application to [free energy calculations, 243, 599-601 protein force field, 243, 595-598 anti-hapten monoclonal antibodies,... 

Remove the slide and apply an antibody for one of the haptens onto the slide and incubate for approximately 15 min in a humidity chamber/box (see Note 20) at room temperature. If the first hapten to visualize is DNP, anti-DNP antibody is applied. For a direct detection with the use of an enzyme-conjugated anti-hapten antibody, skip steps 2 and 3. 

Common haptens used for labeling DNA probes for BISH assays are biotin, DIG, DNP, FITC, and Texas Red. Based on the size of your DNA targets, you may choose from a direct detection or an indirect detection for BISH assays. In general, an indirect detection system can provide better sensitivity compared to a direct detection system. For an indirect detection, you need to select a combination of two antibodies raised with two different animal species, such as a mouse anti-DIG antibody and a rabbit anti-DNP antibody, so that enzyme-labeled anti-mouse antibody and anti-rabbit antibody can be applied for signal detection. If a direct BISH detection is going to be applied, anti-hapten antibodies raised in the same animal species that are labeled with either AP or HRP enzyme molecules... 

The invention of B cell hybridoma technology (K3) has allowed the generation of various kinds of useful antibodies, even very minor or rare antibody species elicited in serum by the conventional procedure, as a pure immunochemical reagent in almost unlimited amounts. Among such new-generation monoclonal antibodies, anti-idiotype antibodies and anti-immune complex (anti-metatype) antibodies have been successfully introduced as key reagents enabling noncompetitive hapten immunoassays (Fig. 11). 

Fig. 11. Schematic representation of the binding properties of anti-idiotype antibodies and antimetatype antibodies. V, hapten a-Id, a-type anti-idiotype antibody /i-Id, /i-type anti-idiotype antibody Met, anti-metatype antibody anti-C-region, antibodies recognizing the constant region of a primary antibody (recognizing isotype or allotype).

Fig. 12. Noncompetitive hapten immunoassay procedures (A and B) using a combination of the a-type and j6-type anti-idiotype antibodies, each recognizing the framework and paratope of the anti-hapten antibody. Anti-hap, anti-hapten antibody (primary antibody) a-Id, a-type anti-idiotype antibody /J-Id, /i-type anti-idiotype antibody S, signal-generating group B, biotin SA, streptavidin.

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. 

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). 

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 ... 

Allen, D., Simon, T., Sablitzky, F., Rajewsky, K., Cumano, A. (1988). Antibody engineering for the analysis of affinity maturation of an anti-hapten response. EMBO J. 7, 1995-2001. 

Barbas III, C. F., Amberg, W., Simoncsitis, A., Jones, T. M., and Lemer, R. A. (1993) Selection of human anti-hapten antibodies from semisynthetic libraries. Gene 137, 57-62. 

Chromogenic Visualization of Dual-Color FISH Signals with Anti-Hapten Antibodies... 

Figure 4. A detection method for chromogenic visualization of dual-color FISH signals with anti-hapten antibody. After FISH-staining with two different fluorochromes such as FITC and Texas Red (a and inset photo a HER2 FISH analysis in breast carcinoma), archival tissue can be immunohistochemically detected using the corresponding anti-fluorochrome antibodies and two different chromogens (b and inset photo b HER2 CISH analysis in the same lesion as in the upper photo).

Altmann KG (1993) Effect of cationization on anti-hapten antibody response in sheep and mice. Immunol Cell Biology 71 517-525... 

Bound fraction Antigen/competitor present as a complex, attached to the corresponding antibody the fi-action of the reaction mixture which contains the antigen-antibody complex Carrier protein Large non-immunogenic protein that, when coupled to a hapten, induces production of anti-hapten antibodies in an animal Competitive assay Immunoassay based on the principle of competition between the analyte in the sample ( unknown ) and competitor Competitor Antigen derivative that is modified (i.e., labelled, coupled to a large protein) and serves for indirect detection of the analsde in competitive immunoassays... 

Fig. 7. Standard immunization protocol for producing anti-hapten monoclonal antibodies...

One of the 6 lysyl residues (residue 97) of lysozyme is near the active site. Conjugation of a hapten to this group does not affect the activity of the enzyme, but incubation of the enzyme-hapten conjugate with the specific anti-hapten antibody prior to the addition of the bulky substrate, produces serious inhibition. The smaller Fab is not as effective for inhibition as complete IgG. 

Enzyme activity can, therefore, be conveniently followed by measuring the formation of NADH at 340 nm. This enzyme is popular in AM-type EIA (Table 10.17). The reaction of anti-hapten antibodies with the hapten attached to the enzyme either inhibits or potentiates the enzyme activity, probably through changes in the conformation of the enzyme. MDase is detectable at 10 " M during a 1-min measurement. (Lysozyme is detectable at 10 M during the same time interval). 

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