Peroxidase streptavidin-conjugated

A very interesting approach was presented recently by Niemeyer et al. [113]. They prepared covalent DNA-streptavidin conjugates to which biotinylated alkaline phosphatase, beta-galactosidase, and horseradish peroxidase, as well as biotinylated anti-mouse and anti-rabbit immunoglobulins, were coupled. Immobilization of DNA-streptavidin conjugates was performed by hybridization with the complementary oligonucleotides, bound to the surface. It was demonstrated... 

Streptavidin-horseradish peroxidase (HP) conjugate (Dako) Store at +4°C. Working solution dilute 1 600 in IX TBS prior to use. 

Chemiluminescent detection is also used, mainly to detect antigen capture on antibody arrays by sequential incubations with biotinylated secondary antibodies and Streptavidin-conjugated horseradish peroxidase (HRP) [61]. Phosphorylation of arrayed kinase substrates in the presence of radiolabeled adenosine triphosphate (ATP) has been detected by autoradiography [62]. 

The separated proteins were probed with streptavidin conjugated to peroxidase. Open and closed arrows indicate the subunit of the plastidic and cytosolic ACCases, respectively. 

Horseradish peroxidase (HRP)-streptavidin conjugate (for transmitted light microscopy). 

Perhaps the most common conjugates of avidin or streptavidin involve attaching enzyme molecules for use in ELISA systems. As in the case of antibody-enzyme conjugation schemes (Chapter 10), by far the most commonly used enzymes for this purpose are horseradish peroxidase and alkaline phosphatase. Other enzymes such as (3-galactosidase and glucose oxidase are used less often, especially with regard to assay tests for clinically important analytes (Chapter 16). 

If p-galactosidasc is used to conjugate with an SMCC-activated avidin or streptavidin, then there is no need to thiolate the enzyme, since it contains sulfhydryls in its native state (Sivakoff and Janes, 1988 Fujiwara et al., 1988). For conjugations using horseradish peroxidase, alkaline phosphatase, or glucose oxidase, however, thiolation is necessary to add the requisite sulfhydryls. 

The CARD approach uses horseradish peroxidase to catalyze the deposition of tyramide molecules that are conjugated to an antihapten immunoglobulin or Streptavidin, which in turn, during the first incubation step, binds to the digoxigenin- or biotin-labeled nucleic acid target. Thus, the number of target sites for the next reaction step is markedly increased. 

In glycoprotein detection systems the carbohydrate portions of proteins are oxidized with sodium metaperiodate to generate aldehydes that can react with hydrazides. A biotin hydrazide is used to attach biotin onto the oxidized carbohydrates and horseradish peroxidase-conjugated streptavidin is used for chemiluminescence-based detection (Glycoprotein Detection Module, Amersham Biosciences, Uppsala, Sweden). 

An example of the immobilization on polystyrene beads is the bead-bed micro-ELISA system of Kitamori et al. [403]. The beads were coated with a capture antibody and loaded into the reaction channel of the ELISA microchip (Scheme 4.92a). Next, solutions containing antigen (Scheme 4.92b), a biotinylated secondary antibody (Scheme 4.92c) and a streptavidin-peroxidase conjugate were introduced into the channel (Scheme 4.92d). Finally, the substrate for peroxidase was continuously... 

Streptavidin (from Streptomyces avidinii) and avidin (from chicken egg) both possess four binding sites for biotin. The biotin molecule is easily conjugated to antibodies and enzymes. In the avidin-biotin complex (ABC) method secondary antibodies are conjugated to biotin and function as links between tissue-bound primary antibodies and an avidin-biotin-peroxidase complex (5). 

In a similar method the labeled streptavidin-biotin (LSAB) method also utilizes a biotinylated secondary antibody that links primary antibodies to a streptavidin-peroxidase conjugate (6). In both methods a single primary antibody is subsequently associated with multiple peroxidase molecules, and because of the large enzyme-to-antibody ratio, a considerable increase in sensitivity is achieved compared to direct peroxidase-conjugate methods. 

In keeping with current trends in immunohistochemistry to develop alternatives to biotin-streptavidin detection methods, a fluorescyl-tyramide amplification system has recently been introduced (FT-CSA). In this procedure peroxidase is associated with a tissue-bound primary antibody by application of a secondary antimouse Ig antibody to which peroxidase has been conjugated. The peroxidase catalyzes the conversion and deposition of fluorescyl-tyramide onto the tissue section. At this point the reaction can be terminated and viewed by fluorescence microscopy, or the signal can be converted to a colorimetric reaction by the sequential application of an anti-fluorsecein antibody conjugated to peroxidase followed by a diaminobenzidine-hydrogen peroxide substrate. 

In most of the assays described the detection of rat or mouse MAb depends on the use of a second antibody reagent specific for F (ab )2 or heavy chain isotype. These second antibodies are detected because they have either a radiolabel ( I), fluorescent tag (fluorescein), or are conjugated to an enzyme (e.g., alkaline phosphatase, peroxidase, or p-galactosidase) either direcdy or indirecdy via a biotin-streptavidin bridge. As examples of these procedures, two altemadve types of methodology, i.e., radioimmunoassay... 

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