Labeled Streptavidin-Biotin

In a similar method the labeled streptavidin-biotin (LSAB) method also utilizes a biotinylated secondary antibody that links primary antibodies to a streptavidin-peroxidase conjugate (6). In both methods a single primary antibody is subsequently associated with multiple peroxidase molecules, and because of the large enzyme-to-antibody ratio, a considerable increase in sensitivity is achieved compared to direct peroxidase-conjugate methods. 

Figure 3. Labeled Streptavidin-Biotin (LSAB) Method.

Other significant improvements were the development of the enzymatic antigen retrieval methods by Huang et al. [18] and the improvement of systems for secondary antibody detection with the introduction of the avidin-biotin-peroxidase complex (ABC) and the labeled streptavidin-biotin complex (LSAB) by Hsu etal. [19-22]. 

ABC avidin-biotin complex, AP alkaline phosphatase, ATS all types of sections, CSA catalyzed signal amplification, EPOS enhanced polymer one step (Dako, Glostrup, DK), HIER heath induced epitope retrieval, HRP horseradish peroxidase, lEnz immunoenzymatic, IMF immunofluorescence LSAB labeled streptavidin-biotin, PAP peroxidase-anti-peroxidase... 

Fig. 34 Functionalisation of bait peptides. I Gold-labelled streptavidin is attached directly. 2 Biotin-labelled anti-FLAG antibody is bound to the FLAG on the peptide (reaction b) and this is used to attach gold-labelled streptavidin (reaction d). Reprinted with permission from Ryadnov and Woolfson [76]. Copyright 2004 American Chemical Society...

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. 

Figure 18.14 NHS-SS-PEG4-biotin can be used to label a primary antibody molecule that has specificity for a protein or interest. Incubation of the biotinylated antibody with a sample, such as a cell lysate, allows the antibody to bind to its target. Capture of the antibody-antigen complex on an immobilized streptavidin reagent effectively isolates the targeted protein from the other proteins in the sample. The disulfide linkage in the spacer arm of the biotin tag permits elution of the immune complex from the streptavidin support using DTT and without using the strong denaturing condition typically required to break the streptavidin-biotin interaction.

Fig. 6.2 Tyramide signal amplification. T is the labeled tyramine and HRP, horse radish peroxidase. The Label can be a lluorochrome or biotin. The lluorochrome can be visualized directly in a fluorescence microscope. Biotin can be visualized via labeled streptavidin...

We (K1) attempted to develop a noncompetitive assay based on the anti-idiotype antibodies for a conjugated bile acid metabolite, ursodeoxycholic acid 7-A-acetyl-glucosaminide (UDCA 7-NAG), which is expected to serve as a diagnostic index for an autoimmune disease, primary biliary cirrhosis. In our assay, the hapten UDCA 7-NAG, a /3-type antibody, and a biotin-labeled a-type antibody were simultaneously added to a microtiter plate coated with an F(ab )2 fragment of a specific anti-UDCA 7-NAG antibody, then incubated at room temperature for 8 h. Bound biotin was then detected with HRP-labeled streptavidin, whose enzyme activity was measured using o-phenylenediamine/H202 as a substrate. This noncompetitive assay system provided a subfemtomole-order sensitivity (detection limit 118 amol) that was 7 times lower than the competitive immunoassay using the same anti-hapten antibody (K2), even with a common colorimetric detection (Fig. 13). Somewhat improved specificity was also obtained namely, better... 

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). 

Figure 10.19 ATP-fueled rotation of fluorescently labeled actin that is attached to the y subunit of the F,-ATPase by a streptavidin-biotin linker. The /3 subunits are immobilized on a microscope slide. [Modified from H. Noji, R. Yasuda, M. Yoshida, and K. Kinosita, Nature 386,299 (1997).]...

For best results, plates should be coated with the IgG fraction of an antiserum, this can be conveniently prepared using caprylic acid precipitation [see Chapter 10 and ref. 15). Where this is not possible, indirect capture may be used, such as antispecies antibody on the plate, or the streptavidin-biotin system. Any indirect capture system must be compatible with the final label, e.g, labeled antigen or different species antisera with no crossreaction with die indirect coating antibody 11. Safety data (from ref. 16)... 

When one or more of the desired antibodies is not available as a direct conjugate and a conjugate cannot be synthesized, indirect methods have to be used. The simplest variation is to use a biotin-labeled antibody as a first layer, and then after washing the cells, incubate with fluorochrome-labeled streptavidin, and additional directly labeled antibodies labeled with complementary fluoro-chromes. The additional streptavidin step does not usually cause difficulty... 

The sections are treated with H202 and then incubated in the primary antibody at a dilution of 1 50. This is followed by sequential incubation in the biotinylated antimouse antibody and streptavidin-biotin-labeled complex. DAB is used for 5 min as the chromogen, and the sections are lightly counterstained with hematoxylin. Positive controls involve the use of the tissue known to express the antigen under study. Negative controls involve the replacement of the primary antibody with the diluent alone or with a non-immune serum. 

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. 

Figure 5 A penicillin-binding activity-based high-throughput SPA assay for the identification of potential inhibitors of the transpeptidase activity of PBPs. A particular PBP of interest is first labeled with biotin. The biotinylated PBP is then mixed with a 3H-labeled P-lactam in 96-well microtiter plates and the reaction mixtures are incubated at room temperature for a period of time. After incubation, the biotionylated PBP-3H-P-lactam complexes are captured by mixing with streptavidin coated SPA beads. The P-lactam-binding activity of the PBP is measured by counting the microtiter plates after mixing with the SPA beads.

Figure 1. Conventional immunohistochemical detection methods. Florseradish peroxidase (HRP) and alkaline phosphatase are commonly employed as enzymes for visualization with chromogen. A The polymer -based method in which dextran polymer is commonly used. B Streptavidin/biotin reaction-based methods including the labeled streptavidin (LSAB) and streptavidin-biotin complex (sABC) methods.

Figure 25.1 Biotinylation of nanodiamond and recognition protocol with horseradish peroxidase-labeled streptavidin. Conditions and reagents (a) BH3 THF, THF, reflux, 72 h (b) APTMS, acetone, room temperature, 16 h (c) biotin, EDC, DMAP, CH2C12, 0°C — room temperature, 65 h.11...

Figure 26. Schematic of the post hybridization detection method. A DNA target solution labeled with biotin is first incubated with the DNA probe functionalized chip. Targets diffuse passively from the solution to the surface where they hybridized with the probes if complementary. A solution containing streptavidin-functionalized magnetic labels is then incubated with the chip. Labels bind through the biotin-streptavidin interaction to where hybridization occurred. DNA hybridization is detected with spintronic transducers.

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