Antibody haptens

HAp coatings, 14 105 HapMap project, 20 839 Hapten-antibody interactions, 9 64 Haptic sensor, for skin conditions, 3 749 Harcros antifoam, commercial defoamer, 3 24 It... 

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

F. V Bright, Multifrequency phase fluorescence study of hapten-antibody complexation, Anal. Chem. 61, 309-313 (1989). 

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... 

Busch et al. studied the applicability of CZE to the examination of hapten-antibody complex formation (11). The catalytic antibodies examined have been used to accelerate a Diels-Alder reaction. Association constants of two hapten-antibody complexes were investigated and compared to the ELISA method. The samples contained buffer, hapten, and antibody. The constants obtained with CZE are a factor of 3-5 larger than those found with the ELISA method. The free-hapten concentration is measured directly this allows confirmation of the stoichiometric model. Because of the poor concentration sensitivity of UV detection, the application of an extended optical path length such as a bubble cell is necessary to obtain reliable binding parameters. 

MHA Busch, HFM Boelens, JC Kraak, H Poppe, AAP Meekel, M Resmini. Critical evaluation of the applicability of capillary zone electrophoresis for the study of hapten-antibody complex formation. J Chromatogr A 744 195-203, 1996. 

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.

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.

Although these assay procedures for steroids were successfully applied to clinical samples, the sensitivity was not extensively improved, as shown by their femtomole range detection limits 2 fmoF/assay for E2, 0.32 nmol/liter (40 fmoF/assay) for progesterone, and 0.4 nmol/liter (8 fmol /assay) for Ei-3G, respectively. Difficulty in obtaining a higher sensitivity would come from the competitive substitution of the bound hapten with the /3 -type antibody and/or imperfect selectivity of the a-type antibody toward the 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. 

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 ... 

M. Franek, M.V. Pouzar and V. Kolar, Enzyme-immunoassays for polychlorinated biphenyls. Structural aspects of hapten-antibody binding, Anal. Chim. Acta, 347 (1997) 163-176. 

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).

Schmidt, D. H., Kaufman, B. M., and Butler, V. P., Persistence of hapten-antibody complexes in the circulation of immunized animals after a single intravenous injection of hapten.. Exp. Med. 139, 278-294 (1974). 

W8. Wehmeyer, K. R., Halsall, H. B., and Heineman, W. R., Electrochemical investigation of hapten. Antibody interactions by differential pulse polarography. Clin. Chem. 28, 1968-1972 (1982). 

Phosphonates and phosphonamidates, which mimic the tetrahedral geometry and anionic character of the transition state for hydrolysis, have proven especially reliable as haptens. Antibodies generated against such compounds readily promote the cleavage of esters and, in a few cases, amides. High levels of stereospecificity are attainable even at chiral centers remote from the reaction site [11]. 

When the stoichiometry of binding exceeds the simplest 1 1 case (e.g., hapten-antibody, which is usually 2 1), we can formulate the equilibrium for the hapten with the divalent (uniform) receptor with invariant identical affinities... 

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

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