Streptavidin-alkaline phosphate

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).

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. 

Bettazzi et al. used an electrochemical low-density DNA array in combination with polymerase chain reaction (PCR) in order to investigate the presence of hazelnut major allergens. Cor a 1.04 and Cor a 1.03, in foodstuff. Unmodified PCR products were captured at the electrode interface via sandwich hybridization with surface-tethered probes and biotinylated signaling probes. The resulting biotinylated hybrids were coupled to a streptavidin-alkaline phosphatase conjugate and then exposed to an a-naphthyl phosphate solution. Differential pulse voltammetry (DPV) was used to detect the a-naphthol signal with detection limits as 0.3 and 0.1 nmol for Cor a 1.03 and Cor 1.04, respectively. The results are comparable with the ones obtained with classical ELISA tests. 

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. 

Streptavidin coupled with phosphatase alkaline (Biospa, Milano, Italy) p-aminophenyl phosphate (PAPP, Universal Sensors, Kinsale-Sandycove, Ireland) 2-(methylamino)ethanol (Aldrich, Milwaukee, USA) bovine serum albumin (BSA), phosphate and tris buffer (Sigma, St Louis, USA). 

Alkaline phosphatase-biotin and oligonucleotide-biotin (herpes simplex vims (HSV) DNA) immobilized onto gold and silver substrates and quartz crystal microbalances coated with avidin and streptavidin p-nitrophenyl phosphate and HSV, respectively [57]... 

A hybridization-based approach has also been developed to quantitate levels of ribozymes in serum using paired complementary oligonucleotide probes, one labeled with biotin and the other with digoxigenin [90]. The annealed triplex is first collected on streptavidin-coated 96-well plates, then an anti-digoxigenin antibody conjugated with alkaline phosphatase is added, followed by addition of the enzyme substrate p-nitrophenyl phosphate, which is cleaved into a soluble colored product that is quantitated by absorbance at 405 nm. 

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