Labeled streptavidin—biotin complex

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]. 

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.

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... 

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.

R.J. Pei, Z.L. Cheng, E.K. Wang, and X.R. Yang, Amplification of antigen-antibody interactions based on biotin labeled protein—streptavidin network complex using impedance spectroscopy. Biosens. Bioelectron. 16, 355—361 (2001). 

HSV in human fibroblasts was detected using biotin-labeled HSV DNA probes, streptavidin-HRP complex, and enhanced CL substrate reagent for HRP [56], The presence of HSV DNA was observed in cells infected with clinical samples known to contain the HSV virus fixed at 48 h postinfection, with a sharp topographical localization and a good preservation of cellular morphology. 

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. 

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. 

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.

E. coli OmpA antibodies, was developed. A biotin-labelled anti-TKSNVYGK monoclonal antibody was first incubated with the P40 solution. After addition of streptavidin, the complex formed between the antibody and residual E. coli OmpA was captured by filtration through a biotinylated nitrocellulose membrane. The membrane was further incubated with a rabbit fluorescein-labelled polyclonal anti-OmpA serum, obtained by... 

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