Immunoassay biotin-avidin system

Matsumura, K. and Fukiya, S. 1992. Indirect competitive enzyme immunoassay for tetrodotoxin using a biotin-avidin system. J. Assoc. Off. Anal. Chem. Int. 75, 883-886. 

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

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). 

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). 

Immunoassay has been widely applied in clinical diagnostics for many years. Diagnostic tests were mostly done using directly coated tubes, particles or microplates coated with antibodies or antigens via adsorption or chemical bonding. However, the test performance could adversely be influenced by the many parameters. Therefore, the avidin-biotin (AB) systems were introduced into the clinical laboratory to replace directly bound antibodies and antigens as solid phase matrices. 

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. 

TTBS is well suited for avidin-biotin systems. For nylon, protein binding agents are recommended. Because nonfat dry milk contains residual biotin that will interfere with the immunoassay, its use must be restricted to the blocking step only. 

Enzyme immunoassay is widely used, both in competitive and non-competitive formats, for the bioanalysis of a broad range of low-molecular-weight compounds and macromolecules. Through the use of fluorogenic substrates and amplification systems such as avidin-biotin, the sensitivity of enzyme immunoassay has been developed to equal or exceed that of radioimmunoas-say.f ° The technique has found particularly wide applicability in the determination of new recombinant proteins, in demonstrating antibody responses to macromolecules, and in the measurement of biomarkers of disease, as well as in diagnostic medicine. 

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). 

Non-competitive solid-phase enzyme immunoassays based on the avidin-biotin system for the determination of antibody activity in sera ... 

T Okumura, et al. Enzyme immunoassay for the drug of antiulcer using avidin biotin system. Chem Pharm Bull 39 1779, 1991. 

A similarly interesting non-covalent complex could be envisioned with the streptavidin or avidin/biotin complex because of its extremely low dissociation constant of 10 15 M. This system has been extensively exploited in immunological techniques, particularly to improve sensitivity and specificity of immunoassays... 

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