Peroxidase, horseradish Probe

L. Alfonta, A.K. Singh, and I. Willner, Liposomes labeled with biotin and horseradish peroxidase a probe for the enhanced amplification of antigen-antibody or oligonucleotide-DNA sensing processes by the precipitation of an insoluble product on electrodes. Anal. Chem. 73, 91-102 (2001). 

Fig. 1 Outline of the scheme for the production of horseradish peroxidase-labeled probes.

Hybridization of Horseradish Peroxidase-Labeled Probes and Detection by Enhanced Chemiluminescence... 

Horseradish peroxidase-labeled probe see Chapter 14) The labeling procedure produces a probe in a single-stranded form it should not be denatured further. 

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

Reduction of the cystamine-labeled oligo using a disulfide reducing agent releases 2-mer-captoethylamine and creates a thiol group for conjugation (Figure 27.6). DNA probes labeled in this manner have been successfully coupled with SPDP-activated alkaline phosphatase (Chapter 26, Sections 1.2 and 2.5), maleimide-activated horseradish peroxidase (HRP) (Chapter 26, Section 1.1), NHS-LC-biotin (Chapter 11, Section 1 and Chapter 27, Section 2.3), and the fluorescent tag AMCA-HPDP (Chapter 9, Section 3 and Chapter 27, Section 2.5). 

The continuous flow method is still necessary when one must use probe methods which respond only relatively slowly to concentration changes. These include pH, Oj-sensitive electrodes, metal-ion selective electrodes,thermistors and thermocouples, " epr and nmr detection. Resonance Raman and absorption spectra have been recorded in a flowing sample a few seconds after mixing horseradish peroxidase and oxidants. In this way spectra of transients (eompounds I and II) can be recorded, and the effext of any photoreduction by the laser minimized. ... 

Probes tagged with an enzyme signal generating system, such as alkaline phosphatase or horseradish peroxidase, are sometimes used in direct assays. Alternatively, enzymes as reporters may be used with biotinylated probes in a direct affinity assay or in an indirect affinity assay. For the former assay, the... 

Tyramide signal amplification (TSA PerkinElmer Life Sciences, Boston) and enzyme-labeled fluorescence (ELF Molecular Probes) are related detection technologies. In the tyramide amplification process, a tyramide-biotin complex is produced by the action of horseradish peroxidase. The complex precipitates near the binding site and accumulates. The complex is detected by the use of streptavidin-Cy3/Cy5. 

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). 

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. 

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