Streptavidin assays

Scheme 9 Schematic illustration of streptavidin assay operation...

Fig. 5.12 On-line continuous-flow monitoring of bioactive compounds using fluorescein-biotin/streptavidin assay. MS instrument Q-ToF2 (Waters) equipped with a Waters Z-spray electrospray (ESI) source. Triplicate injections of (a) biotin-N-succinimidyl ester (m/z 342), (b) N-biotinyl-L-lysine (m/z 373),...

A more recently introduced format is the AlphaScreen assay. The assay principal behind this technology has previously been described above. In the kinase format a biotinylated peptide is bound to a streptavidin donor bead, and a phopshospecific antibody is bound to the acceptor bead. When the substrate is phosphorylated, the beads come in close proximity and a signal is generated. An example using the assay for the detection of inhibitors of serine kinases is presented by Von Leo-prechting [26]. 

Also recently, Liao and collaborators [89] proposed a homogeneous noncompetitive assay of a protein in biological samples based on FRET by using its tryptophan residues as intrinsic donors and its specific fluorescent ligand as the FRET acceptor, which was defined as an analytical FRET probe. To evaluate this method, a naphthylamine derivative, namely /V-biotinyl-/V -(l -naphthylj-ethylene-diamine (BNEDA) 33 was used as an analytical FRET probe for the homogeneous noncompetitive assay of streptavidin. 

A variation on the theme of conventional assay uses both lanthanide-labeled and biotin-labeled single strands to form split probes for sequence of target strands (Figure 12).120 When both of these bind to DNA, the complex binds (via the biotin residue) to a surface functionalized with streptavidin, immobilizing the europium and allowing assay to be carried out. This approach is already very sensitive to DNA sequence, since both sequences must match to permit immobilization of the lanthanide, but can be made even more sensitive by using PCR (the polymerase chain reaction) to enhance the concentration of DNA strands. In this way, initial concentrations corresponding to as few as four million molecules can be detected. This compares very favorably with radioimmunoassay detection limits. 

These techniques have been used to target, detect, or assay glycoproteins in solution or on cell surfaces by using hydrazide-activated enzymes, avidin, or streptavidin (Chapter 23, Section 5) (Bayer and Wilchek, 1990 Bayer et al., 1987a, b, 1990) and to form conjugates with glycoproteins. 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

The increased length of this spacer (24.7 A) provides more efficient interaction potential with avidin or streptavidin probes, possibly increasing the sensitivity of assay systems. The reactions of biotin-LC-hydrazide are identical to those of biotin-hydrazide. 

Biotin modification reagents are widely used to attach a biotin group to proteins or other molecules for subsequent use in avidin, streptavidin, or NeutrAvidin separations or assays. Traditional biotin compounds containing aliphatic or other hydrophobic linker arms are discussed in detail in Chapter 11. In this section, the biotin-PEG compounds exclusively are discussed due to their unique hydrophilic properties, which include low nonspecific binding character and low immunogenicity. 

Several assay designs that use the enhanced sensitivity afforded through biotinylated antibodies have been developed. Most of these systems use conjugates of avidin or streptavidin... 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

A CL ISH assay for simultaneous detection of two different viral DNAs (HSV and CMV DNAs) was developed utilizing both HRP and AP as reporter enzymes [63], A biotinylated HSV DNA probe and a digoxigenin-labeled CMV DNA probe were cohybridized with samples then CL detection of the two probes was performed. The HSV DNA was revealed using a streptavidin-HRP complex... 

As well as fluorescence-based assays, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In a commercial BIA-core system [231] a hydrophobic SPR sensor with an alkane thiol surface was incubated with vesicles of defined size distribution generating a hybrid membrane by fusion of the lipid vesicles with the alkane thiol layer [232]. If the vesicles contain biotinylated lipopeptides their membrane anchoring can be analyzed by incubation with streptavidine. Accordingly, experiments with lipopeptides representing the C-terminal sequence of N-Ras show clear differences between single and double hydrophobic modified peptides in their ability to persist in the lipid layer [233]. 

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. 

Assay for human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood monuclear cells can be performed by PCR followed by detection of PCR products by electrochemiluminescence-labeled oligonucleotide probe [Tris-bipyridine ruthenium (II) complex]. Since one of the PCR primers is biotin-labeled at the 5 end, facile capture of the PCR product-probe complex can be accomplished on streptavidin-conjugated magnetic particles, prior to analysis in an electrochemiluminescence analyzer (S3). 

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