Streptavidin enzyme-labeled

FIGURE 5.6 Schematic representation of the immunosensor based on a Protein A-GEB biocomposite as a transducer, (a) Immobilization of RlgG on the surface via interaction with Protein A, (b) competitive immunoassay using anti-RIgG and biotinylated anti-RIgG, (c) enzyme labeling using HRP-streptavidin and (d) electrochemical enzyme activity determination. (Reprinted from [31] with permission from Elsevier.)... 

Make it visible the fluorophore label can be visualized directly using fluorescent microscopy. The enzyme label must be visualized through an enzyme chromogenic system. Incubate sections with an appropriate enzyme substrate until optimal color develops (see Sect. 2.3). The biotin label can be detected using streptavidin conjugated with an enzyme or fluorophore (see Sect. 6.2.1). 

Tyramide signal amplification (TSA PerkinElmer Life Sciences, Boston) and enzyme-labeled fluorescence (ELF Molecular Probes) are related detection technologies. In the tyramide amplification process, a tyramide-biotin complex is produced by the action of horseradish peroxidase. The complex precipitates near the binding site and accumulates. The complex is detected by the use of streptavidin-Cy3/Cy5. 

For identification the lectins must be labeled. The best labels are biotin (streptavidin-enzyme conjugate) or digoxigenin (anti-digoxigenin antibody-enzyme conjugate), but in the case of Concanavalin A, direct labeling with HRP is possible (see below). 

Enzyme labelling of the detection antibody fill the reservoirs with 15 pL of solution of streptavidin labelled with alkaline phosphatase. Incubate this solution in the micro-channels using 10 multi-loadings so as to ensure the coupling of the biotin moiety of the detection antibody with the ALP-labelled streptavidin. Remove the solution in excess at the end of the pumping process. 

Fig. 9. Enzyme electrochemiluminescence (ECL) immunoassay protocol, (a) Trinitrotoluene (TNT) and dini-trophenyl of haptenylated dextran compete for antibody-binding sites, (b) Antibodies attached to dextran bound to streptavidin-coated paramagnetic beads, (c) ECL detection of enzyme-labelled antibodies magnetically concentrated on electrode. Reprinted from Wilson et al. [68], Copyright (2003), with permission...

Secondary label Biotin Binds avidin-enzyme and streptavidin-enzyme conjugates (activity)... 

PCR-ELISA is a hybrid assay combining PCR as a first step and using ELISA as a detection system for the amplicons (Figure 9.1). The PCR step produces biotinylated amplicons that are bound further to a streptavidin-coated microtiter plate. The amplicons are denatured to produce single-stranded PCR products, and only the biotinylated strand stays in the well of the microtiter plate. A FITC-Iabeled probe is bound to the PCR strand, a step that adds an extra level of specificity to the assay. An enzyme-labeled anti-FITC antibody is used to produce a colorimetric signal that can be measured by an ELISA reader. This method has been applied successfully to the detection of <10 ppm hazelnut in processed foods and food ingredients, which eliminated the potential for cross-reactivity observed in ELISA (Holzhauser et al., 2002). 

Briefly, the procedure consists of the following steps, as schematically outlined in Fig. 3.7 (i) thiolated probe immobilization by chemisorption (Fig. 3.7A) (ii) hybridization with the complementary probe modified with either biotin or digoxigenin (Fig. 3.7B1) (iii) enzyme labeling of the DNA duplex using streptavidin-HRP or anti-DlG-HRP (Fig. 3.7C) and (iv) amperometric determination... 

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