Streptavidin alkaline phosphatase

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... 

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).

Avidin (or streptavidin)-alkaline phosphatase complex Dilution in TBS supplemented with 1% (w/v) BSA as suggested by manufacturer for immunodetection on membranes... 

Incubate the slides in avidin alkaline phosphatase (or streptavidin alkaline phosphatase) diluted 1 100 in TBT. 

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

The sections are treated for 1 hr with streptavidin-alkaline phosphatase in a humid chamber and then washed in PBS. They are developed for 10 min in nitroblue tetra-zolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). After washing in PBS, the sections are treated for 30 min in a double-staining enhancer (Zymed Laboratories, San Francisco, CA) to prevent the first stain from reacting with the second. They are thoroughly rinsed in distilled water followed by PBS. 

An immunohistochemical examination of PSA using polyclonal antibodies by the peroxidase antiperoxidase (PAP) method and by the technique of biotin-streptavidin-alkaline phosphatase has been successfully carried out (Zaviacic et al., 1994). Immunoelectron microscopy in conjunction with the protein A-gold complex can also be used for localizing PSA in human prostate (Sinha et al., 1987). The procedure for immunofluorescence localization of PSA is given below. 

Biotin has served this purpose well in both nucleic acid and antibody probe systems. As well as being easily detected with immunoglobulins specific for biotin, biotin may also be detected non-immunologically with avidin or streptavidin, two proteins which share a marked, highly specific affinity for biotin. The affinity constant for avidin-biotin interactions is approximately 10 - liters/mole, much higher than the range for antigen-antibody interactions which are commonly between 10 -10 liters/mole. Consequently, a vast number of detection complexes composed of avidin or streptavidin bound to a detection system are commercially available (e.g. streptavidin-alkaline phosphatase). 

Biotinylated target protein followed by streptavidin-alkaline phosphatase are used to screen bead library positive beads turned dark upon adding of BCIP substrate. 

Meanwhile, make up the antibody conjugates. To one universal tube, add 5 mL of Buffer B and to another add 5 mL of Buffer C. To the first tube, add 5 pL of streptavidin alkaline phosphatase (for biotin-labeled probes) and 5 pL of anti-dig alkaline phosphatase (for dig-labeled probes). 

A disposable electrochemical competitive assay for detection of IgE was proposed by Papamichael et al. [16]. In this work the IgE antigen was immobilized on the surface of screen-printed electrodes, then a competition step between IgE bound to the electrode surface and IgE in solution for the biotinylated aptamer was left to occur. At this point the streptavidin-alkaline phosphatase conjugate... 

Figure 9.4. Schematic representation of a impedimetric genosensor [sandwich hybridization assay]. Unmodified PGR products [b] were captured at the sensor interface [a] via sandwich hybridization with the surface-tethered probe and a biotinylated signaling probe. The biotinylated hybrid [c] was then coupled with a streptavidin-alkaline phosphatase conjugate [d] and finally exposed to the substrate solution [e]. The biocatalyzed precipitation of an insulating product [f] blocked the electrical communication between the gold surface and the [Fe(CN]6] redox probe [published by Elsevier in Ref. 50].

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