Immunoassay avidin—biotin complex

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... 

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. 

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). 

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). 

Competitive immunoassays based on the avidin-biotin approach have also been described. In this case, the antibody is immobilized on microtiter plates. Wells are incubated with a constant concentration of biotinylated antigen in the presence of different concentrations of standards or the sample. After washing, the ABC complex is added. The formed antibody-biotinylated antigen-ABC sandwich is then detected by the addition of enzyme substrate. The immobilized enzyme-activity is inversely proportional to the concentration of analyte in the sample, resulting in a typical sigmoidal calibration curve. 

Antibody-masking enzyme tag immunoassay Adenosine 5 -monophosphate S-Acetylmercaptosuccinic anhydride Alkaline phosphatase anti-alkaline phosphatase (enzyme-antibody) complex Alkaline phosphatase 5-Aminosalicylic acid Adenosine 5 -triphosphate Aa-Benzoyl-L-arginine ethyl ester (-f-)-Biotin bromoacetyl hydrazide (-b)-Biotin Y-aminocaproic acid A-hydroxy-succinimide ester Bis-diazotized benzidine -Galactosidase (-I- )-Biotin hydrazide (-I- )-Biotin-A-hydroxysuccinimide ester (-I-)-Biotin p-nitrophenyl ester Bridged avidin-biotin (method)... 

Obelin-biotin complex was successfully obtained using succinimide derivatives of biotin, with less than 30% of its bioluminescent activity lost under the synthesis conditions. The biotinylated obelin is a universal label, suitable for any immunoassay through the avidin bridge. As an example, Fig.l gives the scheme of a solid-phase microanalysis of alphafetoproteins in standard human sera and displays the results of this analysis. 

Figure 1 Immune complexes formed in typical PCR immunoassay. The two general configurations for DNA immunoassay that are shown differ in the means of capture. Analyte DNA is assumed to have been amplified with a 5 -biotinylated primer. In the immunocapture approach, biotin provides the means for capture onto an avidin-coated multiwell plate. In the hybridization approach, the analyte is captured by hybridization to an immobilized probe followed by immunoenzymatic detection with an avidin-alkaline phosphatase conjugate.

Antibodies form complexes with their respective antigens (here analytes). These complexes can show a particular strength, which can be quantified by the affinity constant (or equilibrium constant). This affinity constant is about 10 -10 L mol for most analytically useful antibodies. The higher this number, the more stable is the complex. The highest known affinity in the biochemical field is the interaction between avidin (an egg protein) and biotin (vitamin H), for which a value of around 10 L mol has been determined. The affinity constant plays an important role in immunoassays and other immunological techniques. The development of new methods is greatly facilitated if this constant is known. 

---