Streptavidin alkaline phosphatase conjugate

While enzymes may be covalently attached directly to primary probe molecules, as noted above for reasons of reagent versatility, steric factors, and potential signal amplification, indirect detection systems appear to be the more popular. Consequently, enzyme-probe conjugates are typically complexes of a desired enzyme marker and a secondary level probe that is, a probe molecule that can specifically identify a primary level probe molecule, such as an alkaline phosphatase-streptavidin conjugate can identify a biotinylated nucleic acid probe by virtue of the binding affinity between streptavidin and biotin. Other examples of enzyme-probe systems are given in the preceding section on direct and indirect detection systems. 

Fig. 4. Schematic diagram of the steps in the automated PCR/OLA procedure performed with a robotic workstation. The assay contains three steps (1) DNA target amplification (2) analysis of target nucleotide sequences with biotin (B)-labeled and digoxigenin (D)-labeled oligonucleotide probes and T4 DNA ligase (L) and (3) capture of the biotin-labeled probes on streptavidin (SA)-coated microtiter wells and analysis for covalently linked digoxigenin by using an ELISA procedure with alkaline phosphatase (AP)-conjugated antidigoxigenin (aD) antibodies and a substrate (S). Reprinted with the permission of Nickerson el al. (N2) and the Proc. Natl. Acad. Sci. (U.SA.).

An enzyme-amplified detection scheme, based on tire coupling of a streptavidin-alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalysed the hydrolysis of the elecn oiiractive a-naphthyl phosphate to a-naphtlrol this product is elecU oactive and has been detected by means of differential... 

The on-bead assay was conducted according to Scheme 3.19, which shows the chain of events, which leads to a colorimetric response, when an oligosaccharide binds effectively to the B. purpurea lectin. The lectin was covalently linked to biotin, a small molecule with an extremely high affinity for streptavidin. The bead-lectin-biotin conjugates were then exposed to streptavidin, linked to the enzyme alkaline phosphatase. Alkaline phosphatase hydrolyses phosphate esters [e.g., 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 110]. When the 5-bromo-4-chloro-3-hydroxyindole (111) is released, in the presence of nitro blue tetrazolium (NBT), it forms a dark purple, insoluble dye, thus staining beads where there was a favorable binding interaction. 

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... 

Figure 14.8. Enzymatically-amplified TR-FIA ofa-fetoprotein (AFP), using streptavidin-alkaline phosphatase conjugate (SA-ALP) and 5-lluorosalicyl phosphate (FSAP) to generate 5 fluorosalicylic acid (FSA). The FSA comhines with terbium-EDTA to form a bright, fluorescent complex. (Adapted from Ref. 83 with permission.) (01992, American Chemical Society).

Streptavidin (Boehringer Mannheim, Mannheim, Germany) blocking buffer (1 pg/mL). Biotin-conjugated alkaline phosphatase (Boehringer Mannheim), in blocking buffer, 5 pg/mL. 

Streptavidin labeled with 125I or conjugated to alkaline phosphatase or to fluorescein... 

Detection of the sites of hybridization-dependent binding of biotinylated probe to the filters is most readily conducted with commercially available kits. Favorable results have been obtained with the BluGene Nonradioactive Nucleic Acid Detection System from BRL. Follow the manufacturer s instructions when carrying out the following steps. After washing, sequentially expose the filters to streptavidin and biotinylated alkaline phosphatase (or to a conjugate of these two proteins) This causes the immobilization of alkaline phosphatase at sites of positive hybridization... 

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. 

Screening of an Encoded Peptidomimetic Library with Streptavidin-Alkaline Phosphatase Conjugate... 

During this step, biotin sites on bound tyramine act as additional binding sites for antibiotin enzyme. An additional round of signal amplification can be achieved by using biotinyltyramide and streptavidin conjugated to alkaline phosphatase (Yang et al., 1999). 

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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