ELISA using avidin-biotin

Using the ELISA (with avidin-biotin bridges) and ELISPOT methods a stimulating effect of selected probiotic bacteria (Bifidobacterium longum, B animalis, Lactobacillus easel, L. salivarius) on the immune system of the rat and mouse has been demonstrated (higher level of specific as well as total IgGa and IgA content) (Nagy et al., 2002). 

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). 

A rapid avidin/biotin ELISA has been developed for the determination of bovine somatotropin in blood and milk (150). The method uses affinity-purified polyclonal antisera raised in rabbits to immobilize bovine somatotropin from... 

We perceived the need for sensitive assays that do not rely on the use of radioisotopes or extensive analytical methodology and that could accurately detect protein-bound acetaminophen in biological fluids in the presence of unbound acetaminophen. To this end, we recently developed sensitive avidin biotin-amplified ELISA (A-B ELISA) and particle concentration fluorescence immunoassays (PCFIA) which use antiserum specific for the major acetaminophen-protein adduct associated with toxicity (13-16). These assays are new tools to study the relation between formation of the 3-Cys-A protein adduct and acetaminophen-induced toxicity. In this report we review how these assays were developed, validated in laboratory animals, and used to quantify 3-Cys-A protein adduct formation in human acetaminophen overdose patients. 

Foiles, P. G. Trushin, N. Castonguay, A. Measurement of 0-6-methyldeoxyguanosine in DNA mefliylated by the tobacco-specific carcinogen 4-methylnitrosamino-l-3-pyridyl-l-butanone using a biotin-avidin enzyme-linked immunosorbent assay ELISA. Carcinogenesis (Lond), 6 989-94. 1985. 

Antibodies have been produced to both retronecine and monocrotaline and detected using an avidin-biotin antibody. Retronecine was obtained from the hydrolysis of monocrotaline, succinylated and coupled to bovine serum albumin or ovalbumin. Recently an ELISA lateral flow device (dipstick test) based on gold colloidal polyclonal antibodies has been developed for jacobine and lycopsamine in honey and animal feed [59]. 

A number of additional strategies for signal amplification in ELISAs have been developed. One example is the use of secondary antibodies conjugated to biotin, a molecule that has very strong affinity (>10 1 mol ) for the egg yolk protein avidin. After application of the secondary antibodies and washing to remove unbound reagents, the substrate is flooded with an excess of enzyme-conjugated avidin. Not only is the interaction between avidin and biotin... 

Several established protocols have been adapted for 96-well plate readers including catalase, hyaluronidase, acetylcholinesterase, protein phosphatases and membrane-bound ATPases (22-26). In several instances these have involved novel protocols that are well suited to the ELISA format. For example, a sensitive, rapid microtitre-based assay for hyaluronidase activity was described by Frost and Stem (23). The free carboxyl groups of hyaluronan are biotinylated in a one-step reaction using biotin-hydrazide. This substrate is then covalently coupled to a 96-well microtitre plate. At the completion of the enzyme reaction, residual substrate is detected with an avidin-peroxidase reaction that can be read in a standard ELISA plate reader. Because the substrate is covalently bound to the microtitre plate, artefacts such as pH-dependent displacement of the biotinylated substrate do not occur. The sensitivity permits rapid measurement of hyaluronidase activity from cultured cells and biological samples, with an interassay variation of less than 5%. 

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