Biotin-avidin detection

Based apparently on the same principle as described above, suitable but rather expensive kits exploiting monovalent Fab fragments to block endogenous tissue immunoglobulins have also been developed commercially, such as Mouse-on-Mouse (M.O.M. ) Kits by Vector Laboratories (http //www.vectorlabs.com/). Three Vector M.O.M. kits are available. These kits use the same blocking technology and biotin-avidin detection format, but offer a choice of using either an enzyme-based or fluorescent-based visualization method. 

Wood G, et al. Suppression of Endogenous Avidin-Binding Activity in Tissues and Its Relevance to Biotin-Avidin Detection Systems. Journal of Histochemistry and Cytochemistry 2981 29 1196-204. 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

The technique described here is for use with monoclonal primary antibodies of mouse origin, but can easily be adapted for use with polyclonal antibodies from other species (i.e., rabbit). This method uses a secondary biotin-labeled antibody and a detection system that employs a biotin-avidin horseradish peroxidase complex linker step, the so-called ABC (avidin-biotin complex) detection system (5) (see Chapter 25). In this detection system, avidin acts as a bridge between the biotinylated secondary antibody and a biotin-labeled peroxidase enzyme. The anchored enzyme, in the presence of H2O2 can then convert the substrate, diaminobenzidine, to a brown or black reaction product that is easily identifiable in the tissue section. 

One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin—avidin or biotin—streptavidin complexes. The avidin— biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 13) (Savage et al., 1992). 

A cocktail (1 1) of two monoclonal anticyclin Dl/bc 1-1 antibodies were used P2D11F11 (diluted 1 40) and 5D4 (diluted 1 100) these two antibodies can be obtained from Vecta Laboratories, Inc., Burlingame, CA, and Immunotech, Westbrook, ME, respectively. The avidin-biotin immunoperoxidase detection system employing DAB is used as the chromogen (Ventana Biotek). After counterstaining in dilute Mayer s hematoxylin, the sections are dehydrated and mounted in Permount. 

Warnke, R., and Levy, R. 1980. Detection of T and B cell antigens with hybridoma monoclonal antibodies. A biotin-avidin horseradish peroxidase method. J. Histochem. Cytochem. 8 771-776. 

Fig. 18.2. Biosensor scheme describing the various steps involved in the immunoassay using ITO-poly(pyrrole-biotin)-coated optical fibers for the detection of anti-cholera toxin B subunit using the biotin-avidin immobilization technique.

Jeon, M., J. Kim, K.-J. Paeng, et al. 2008. Biotin-avidin mediated competitive enzyme-linked immunosorbent assay to detect residues of tetracyclines in milk. Microchem. J. 88 26-31. 

Detection and therapy of lesions with biotin/avidin polymer conjugates for amplification of targeting. [G. L. Griffiths, PCT Int. Appl. 9423759 1994 Chem. Abstr. 122 38823] [ 1841],... 

Immunohistochemical staining can be direct or indirect. Direct immunohis-tochemical staining methods utilize only a primary antibody, which may be conjugated to horseradish peroxidase, biotin, alkaline phosphatase, or other chromogens. In the case of biotin-labeled primary antibodies, avidin or strep-tavidin linked to peroxidase binds to the biotin allowing detection of reactivity of the test antibody with the tissue. Indirect immunohistochemical staining methods utilize secondary, tertiary, or even quaternary antibodies, any of which may be linked either to biotin or enzyme (e.g., peroxidase). 

In terms of biosensing applications using such layers, again cholera toxin detection on a porous silicon substrate [85] has been reported. Also biotin-avidin interaction by QCM [86], glutamate detection [87], as well as protein membrane interactions [88, 89] have been studied. 

Probe Detection. There are a number of different protocols for the detection of hybridized probe and these are summarized in Table 2. The choice of detection system will be dictated by your choice of probe label, (biotin or dig), and whether you are carrying out single or double hybridizations. For single hybridizations with biotin-labeled probes, we favor the avidin-detection protocol (see Subheading 3.8.1.), and for double hybridizations with biotin- and dig-labeled probes simultaneously, the avidin/anti-dig protocol (see Subheading 3.8.3.). 

Biotin deficiency occurs when large amounts of raw egg white are consumed. Egg white contains avidin, a protein (M.W. 70,000) that binds biotin strongly and specifically, preventing its absorption from the intestine. Because of the tight binding and specificity of biotin, avidin-labeled probes have been used to detect proteins and nucleic acids to which biotin has been covalently attached ( biotinylated molecules). Avidin is a homotetramer. Each subunit contains 128 amino acids and binds one molecule of biotin. The affinity of avidin for biotin is abolished by heat and other denaturants. Biotin deficiency can result from sterilization of the intestine by antibiotics and from administration of biotin analogues. 

FIGURE 1.8 (A) Direct biotin-avidin method. The primary antibody is linked to biotin (B) an avidin-peroxidase-conjugate (A-Px) is then added. (B) Indirect biotin-avidin method. Used for monoclonal antibodies, the primary antibody is not conjugated its localization is detected by a biotinylated secondary antibody. Boxed asterisk represents antigen determinant on primary antibody. Px, peroxidase label A, avidin B, biotin. From Taylor CR, Cote RJ, eds. Immunomicroscopy A Diagnostic Tool for the Surgical Pathologist. 3rd ed. Philadelphia Elsevier 2005 21. 

Avidin was the first commercially available recombinant protein (1997) to be derived from a transgenic system (corn seed) for use in medical applications [285, 286, 291]. As it forms strong, non-covalent bonds with biotin, avidin is used in medical and biochemical diagnostic kits for the detection of biotin-containing proteins and nucleic acids. Transgenic corn which accumulated avidin was developed jointly by... 

Figure 36. Biotin-avidin system for detection and purification of oligonucleotides. Modified from [277] Examples for direct ligand incorporation.

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