Labelled streptavidin-biotin method

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.

Figure 2. Coronin 1C in melanoma. A) Immunohistochemical staining of the indicated tissue samples for Coronin 1C. S m sections from melanoma tumors were stained with a new Coronin 1C specific mAb (Roadcap and Bear, unpublished reagent) for 30min at 37°C with a steam heat-induced epitope retrieval, using the streptavidin/biotin method with an alkaline phosphatase label and Permanent Red (Dako) as the chromogen and then stained with hematoxylin. B) qRT-PCR analysis of the indicated melanoma cell lines treated with the Erk inhibitor U0126 or vehicle control for 24 hours.Dotted line indicates lx threshold.

There are several ways of introducing alkaline phosphatase labeled reagents, essentially paralleling those methods used with horseradish peroxidase. Ongoing improvements in polymer-based methods (discussed in the following section) are so dramatic that it appears likely that these methods will supersede PAP and streptavidin biotin methods as the primary method as well as for double stains. However, a brief description of special alkaline phosphatase applications follows. 

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]. 

This method, either standard or elite (increased molar ratio), remains the forefront of much of the immunocytochemistry being performed today, and will be the main subject of this chapter. There are new methods, though, that are being used with increased frequency, such as the labeled-avidin binding method, sometimes called the streptavidin-binding method, and a newer catalyzed amplification method that uses avidin, biotin, peroxidase, and a biotinyl tyramide to achieve even more sensitivity. These methods will be discussed at the end of this chapter. 

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... 

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 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.

Fig. 1 Diagram illustrating the molecular interactions of direct immunofluorescent labeling method. The fluorochrome-labeled antibody reacts with the cell or tissue antigen in a one-step staining procedure (a). Alternatively, the primary antibody, labeled with biotin, can be used with a secondary fluorochrome-streptavidin reagent to increase signal strength (b).

Fig. 13. Schematic representation of direct labelling methods using (A) primary antibody coupled to gold or (B) Fab fragment coupled to gold and indirect labelling methods using (C) protein A-gold, (D) secondary antibody coupled to gold or (E) the streptavidin-biotin system, showing clearly the difference between resolution and sensitivity.

Huhtinen P, Soukka T, Lovgren T et al (2004) Immunoassay of total prostate-specific antigen using europium(III) nanoparticle labels and streptavidin-biotin technology. J Immunol Methods 294 111-122... 

GBPs or microorganisms can be pre-labeled with any appropriate fluorescent dye or biotin, which can be further detected with fluorescently-labeled streptavidin. The following method is based on biotinylated plant lectins. The expected results are shown in Fig. 2. 

The reagent also has been used in a unique tRNA-mediated method of labeling proteins with biotin for nonradioactive detection of cell-free translation products (Kurzchalia et al., 1988), in creating one- and two-step noncompetitive avidin-biotin immunoassays (Vilja, 1991), for immobilizing streptavidin onto solid surfaces using biotinylated carriers with subsequent use in a protein avidin-biotin capture system (Suter and Butler, 1986), and for the detection of DNA on nitrocellulose blots (Leary et al., 1983). 

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