Immunoassay labeled avidin—biotin system

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 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. 

Figure 8. Principle of an immunoassay using the biotin-avidin system for labeling antibodies...

Two-site immunometric or sandwich assays that made use of two or more antibodies directed at different parts of the PRL molecule were next to be developed. As with other two-site IRMA assays, the capture antibody is attached to a solid phase separation system and the second or signal antibody is labeled with a detection molecule (e.g., radio-isotope, enzyme,fluorophor, or chemiluminescence tag ). In some assays, the capture antibody is attached to the wall of test tubes, plastic beads, microtiter plates, ferromagnetic particles, or glass-fiber paper. Other assays have used the strep-avidin approach that couples biotin to the signal antibody with avidin linked to a solid phase. Most of the current immunometric assays for PRL have been adapted to fully automated immunoassay systems. Compared with the older traditional RIA methods, these automated immunometric assays for PRL generally achieve lower detection limits (0.2 to 1.0 ig/L) and improved precision (interlaboratory coefficients of variation of <8% at all concentrations), and have superior specificity (<0.05% crossreactivity with GH). 

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