Probe haptenation

Bisulfite modification of cytosine residues also can be used to add permanently a sulfone group to the C-6 position. In this scheme, the sulfone functions as a hapten recognizable by specific anti-sulfone antibodies. At high concentrations of bisulfite and in the presence of methyl-hydroxylamine, cytosines are transformed into N4-methoxy-5,6-dihydrocytosine-6-sulphonate derivatives (Herzberg, 1984 Nur et al., 1989). Labeled antibodies can then be used to detect the hybridization of such probes. 

Hopman, A.H.N., Wiegant, J., Tesser, G.I., and Van Duijn, R (1986) A nonradioactive in situ hybridization method based on mercurated nucleic acid probes and sulfhydryl-hapten hgands. Nucleic Acids Res. 14, 6471-6488. 

At the EM level, detection usually involves using a probe (oligonucleotide) in which a hapten has been incorporated. Incorporation of the hapten does not interfere with the hybridization of the complementary sequences. The next step is the binding of a reporter (may be an antibody) to the hapten. The reporter is then subjected to a binding molecule (may be a secondary antibody) that is coupled with an electron-dense material such as colloidal gold for visualization. Nonetheless, the many affinity-detection and immunodetection systems developed for immuno-cytochemistry may now with ingenuity be applied to molecular biology at the EM level. 

Figure 14.3. Distinction between homogeneous and heterogeneous immunoassay formats. Haptenic analytes are indicated as triangles, and conjugated fluorescent probes are indicated by the letter F. In this hypothetical depiction, the homogeneous immunoassay is quantitated in the original reaction mixture. The heterogeneous immunoassay requires removal of unreacted tracer, further addition of reagents such as an enzyme to release a fluorescent molecule F, followed by quantitation.

Figure 14.4. Diagram of three basic immunoassay formats, (a) Common competitive format (b) competitive assay with immobilized antigens bound to a carrier protein (immunometric assay) (c) two-site immunometric assays. Haptenic analytes are indicated as triangles, whereas larger-molecular-weight analytes are shown as teardrop shapes. Conjugated fluorescent probes are denoted by the letter "F. ...

AP- and/or horseradish peroxidase (HRP)-conjugated antibodies for the haptens used for labeling probes (a direct detection method) or unconjugated antibodies for the haptens and AP- and/or HRP-labeled anti-species antibodies (an indirect detection method) (Vector Laboratories, Inc., Bnrlingame, CA, USA) (see Note 8). 

Hapten-labeled DNA probes for FISH assays can be used for BISH assays and those probes can be purchased from various vendors. If commercial hapten-labeled DNA probes are not available, DNA probes can be labeled with haptens by nick translation or by PCR in a laboratory. Nick translation kit can be utilized for labeling DNAprobes with hapten (10976776001, Roche Applied Science, Germany). New probes must be analyzed for the specificity by FISH or BISH assays using a comparative genomic hybridization metaphase control slide. 

ZytoVision ZytoDot2C SPEC probes are labeled with DIG and DNP haptens and the probe hybridization sites can be visualized with a BISH detection kit including anti-DIG and anti-DNP antibodies. 

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... 

Probes can be differently labeled with hapten labels, for example carboxyfluorescein (6-FAM), digoxigenin (DIG) and biotin can be bound to LNA oligos. The choice of probe label depends on experimental design and the techniques available in the laboratory. The hapten label provides a template for crucial signal amplification since the FITC label on the oligo itself is not sufficient to allow detection in standard epifluorescence. In this study, the fluorescence signal was obtained with the TSA-FITC substrate, which allowed detection of miR-21 and miR-205. 

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.

Sugars substituted by a functionalized fluoroalkyl chain have been used to glycosylate proteins, such as albumin (BSA, HSA) and haptenes. In the latter case, the fluorine atoms can be used as probes in F NMR to determine quantitatively the bonding of the haptene. " ... 

Haugland, R. P. (1996) Biotin and Haptens, in Handbook of Fluorescent Probes and Research Chemicals, 6th ed. (Spence, M, ed), Molecular Probes, Inc., Eugene, OR, Chapter 4... 

Essentially, the CARD protocol is based on the deposition of haptenized tyramide molecules in the vicinity of hybridized probes catalyzed by horseradish peroxidase. The success of this technique depends on the integrity of target mRNA in sections and the ability of the probe to penetrate the sections and hybridize with mRNAs. Another requirement is an efficient reporter system capable of revealing low numbers of probe-mRNA hybrids per cell accompanied by low background staining. 

Synthesis of Type 2 Human Blood Group Antigenic Determinants. The H, X and Y Haptens and Variations of the H Type 2 Determinants as Probes for the Combining Site of the Lectin 1 of Ulex europaeus, O. Hindsgaul, T. Norberg, J. LePendu, and R. U. Lemieux, Carbohydr. Res., 109 (1982) 109-142. 

In 1996, Gauglitz and coworkers coated surfaces with various amino-and carboxy-substituted polymers [198], The polymers tested were branched poly-(ethyleneimine), a,co-amino-functionalized PEG, chitosan, poly(acrylamide-co-acrylic acid) and an amino-modified dextran. The amino-substituted polymers were immobilized on glass by first immobilizing an aminosilane, followed by succinic anhydride/A-hydroxysuccinimide linker chemistry. Poly(acrylamide-co-acrylic acid) was directly coupled to an aminosilanized surface. When probed with 1 mg mL 1 ovalbumin solution, nonspecific adsorption was lowest for the dextran derivative. Notably, nonspecific adsorption increased in most cases when a hydrophobic hapten (atrazine) was coupled to the polymer-modified surface. 

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