Labeled avidin-biotin

In situ hybridization techniques are used to subtype the papilloma virus that may be found in premalignant lesions in uterine cervix. Most of the techniques use nonradioactively labeled avidin-biotin probes. A number of specific biotin-labeled probe cocktails are available for HPV subtype identification (e.g., HPV 6-l l, 16-18, 31-33-35). Some of these techniques use chemiluminescent components to enhance the sensitivity of HPV subtype detection (H5). 

Elias, J., Margiotta, M., and Gabore, D. (1989) Sensitivity and detection efficiency of the peroxidase antiperoxidase (PAP), avidin-biotin complex (ABC), and the peroxidase-labeled avidin-biotin (LAB) methods. Am. J. Clin. Pathol. 92,62. 

Simultaneous, double immunostaining of two antigens in single cells in sections of formalin-fixed and paraffin-embedded archival tissues can be carried out. This is accomplished by using microwave heating to detect otherwise undetectable nuclear antigens, followed by the labeled avidin-biotin (LSAB) procedure and the alkaline phosphatase (APAAP) protocol to detect cytoplasmic or membranous antigens (Bohle et al 1997). 

Sections (4 xm thick) of formalin-fixed and paraffin-embedded ovarian tissue are mounted on silane-coated slides and air-dried for 24hr at 3°C (Davidson et al., 2000). They are deparaffinized, rehydrated, placed in 0.01 M sodium citrate buffer (pH 6.0), and heated twice for 5 min each in a microwave oven. 2H5 antibody (PharMingen, Becton Dickinson, San Jose, CA) is used at a concentration of 2 p,g/ml to detect sialyl Lex antigen. Staining is performed with labeled avidin-biotin. Negative controls consist of the exclusion of the primary antibody, while positive controls consist of carcinomas that have been shown to be immunoreactive for the antigen in earlier studies. 

The avidin-biotin system was developed for detecting antigens at the electron microscope level (Heitzmann and Richards, 1974). Later Heggeness and Ash (1977) proposed the use of this system for fluorescence immunohistochemistry. Guesdon et al. (1979) proposed a variety of labeled avidin-biotin methods which were further supported by Warnke and Levy (1980). The avidin-biotin methods used today are similar to the system described by Hsu et al. (1981). This system is a significant improvement over the previous immuno-histochemical techniques. The problem of endogenous biotin is discussed on page 98. 

FIGURE 3.1.2 The principle of antigen determination by the immune methods with avidin-biotin signal amplification, (a) Labeled avidin-biotin assay (LAB assay) (b) Bridged avidin-biotin assay (BRAB assay) and (c) Avidin-biotin complex method (ABC assay). (From jQdrychowski, L., Pol. J. FoodNutr. Sci., 12/53(SI 2), 32, 2003b. With permission.)... 

Isotope Dilution Assay. An isotope dilution assay for biotin, based on the high affinity of avidin for the ureido group of biotin, compares the binding of radioactive biotin and nonradio active biotin with avidin. This method is sensitive to a level of 1—10 ng biotin (82—84), and the radiotracers typically used are p C]biotin (83), [3H]biotin (84,85) or an I-labeled biotin derivative (86). A variation of this approach uses I-labeled avidin (87) for the assay. 

Agiamamioti K, Triantis T, Papadopoulos K, Scorilas A (2006) 10-(2-Biotinyloxyethyl)-9-acridone a novel fluorescent label for (strept)avidin-biotin based assays. J Photoch Photobio A 181 126-131... 

Figure 8.1 The results of IHC of two experiments using Dynabeads (Dynal, New York, NY) coated with biotinylated anti-mouse IgG (first experiment) and protein S-100 (second experiment), (a) Positive control showing red color (S-100) localized in the melanoma cells, (b) Strong positive red color circles all beads coated with biotinylated anti-mouse antibody after the heating AR treatment (first experiment), (c) Using the heating AR treatment, S-100-coated polymer beads show positive red color around the beads as circles (second experiment), (d) Negative control of the first experiment. No red color could be seen for polymer beads (arrows) that had been treated with exactly the same protocol as that of slide (b), but omitting the avidin-biotin-peroxidase (label). Bar = 50pm. Reproduced with permission from Shi et al., J. Histochem. Cytochem. 2005 53 1167-1170. See color insert.

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. 

Figure 7.21 Dendrimers that are fluorescently labeled as well as biotinylated create enhanced detection reagents for use in (strept)avidin-biotin-based assays. Large complexes containing multiple fluorescent dendrimers can bind to antigens and form a highly sensitive detection system that exceeds the detection capability of fluorescently labeled antibodies.

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). 

A common application for (strept)avidin-biotin chemistry is in immunoassays. The specificity of antibody molecules provides the targeting capability to recognize and bind particular antigen molecules. If there are biotin labels on the antibody, it creates multiple sites for the binding of (strept)avidin. If (strept)avidin is in turn labeled with an enzyme, fluorophore, etc., then a very sensitive antigen detection system is created. The potential for more than one labeled (strept)avidin to become attached to each antibody through its multiple biotinylation sites is the key to dramatic increases in assay sensitivity over that obtained through the use of antibodies directly labeled with a detectable tag. 

Similar techniques can be used to devise (strept)avidin-biotin assay systems for detection of nucleic acid hybridization. DNA probes labeled with biotin can be detected after they bind... 

Other fluorescent probes also may be used to label (strept)avidin molecules for detection of biotinylated targeting molecules. Chapter 9 reviews many additional fluorescent labels, such as quantum dots, lanthanide chelates, and cyanine dye derivatives, all of which may be used in similar protocols to create detection conjugates for (strept)avidin-biotin-based assays. 

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