Labeled avidin binding

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. 

Key words Avidin, Biotin, Avidin-biotin complex (ABC), Labeled avidin binding (LAB), Biotinylated tyramide, TBS buffer, DAB chromogen... 

Next, a labeled avidin binds to the biotin. This method has three incubation steps... 

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. 

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. 

The presence of biotin labels on an antibody molecule provides multiple sites for the binding of avidin or streptavidin. If the biotin binding protein is in turn labeled with an enzyme, fluorophore, etc., then a very sensitive detection system is created. The potential for more than one labeled avidin to become attached to each antibody through its multiple biotinylation sites provides an increase in detectability over antibodies directly labeled with a detectable tag. 

The procedure for SPDP activation of phycobiliproteins can be found in Chapter 8, Section 1.7. Reacting the SPDP-activated phycobiliprotein with SH-labeled avidin at a molar ratio of 2 1 will result in highly fluorescent biotin-binding probes. 

The low-molecular-weight vitamin biotin is easily conjugated to antibodies and enzyme markers. Up to 150 biotin molecules can be attached to one antibody molecule, and the strong affinity of the biotin for the glycoprotein avidin allows its use as complex-ing secondary reagents. Biotin labeling of the primary (direct) or secondary (indirect) antibody can be used in the avidin-biotin methods. In the labeled avidin method the tracer is attached directly to the avidin molecule. In the avidin-biotin bridge method a biotinylated enzyme such as peroxidase is allowed to bind after attachment of avidin to the biotin-labeled antibody. 

Secondary label Biotin Binds avidin-enzyme and streptavidin-enzyme conjugates (activity)... 

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