Biotinylation biotin targeting assay

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. 

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. 

Similarly, Coutlee et al. (1989a) developed a sandwich assay in which (i) hybrids formed in solution between the biotinylated hapten probe and the target were collected by anti-biotin antibodies immobilized in wells of a microtiter plate and (ii) detected by /3-galactosi-dase-Fab fragment (of monoclonal antibody to RNA DNA) by conversion of a substrate to a fluorescent product. Optimum conditions included hybridization for 16 h at 75°C in 2 X SSC, 10 mM HEPES, using 0.1 xg/ml probe with 7% biotinylation. 

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