Enzyme-linked biotinylated

Valdivieso-Garcia, A. Riche, E. Abubakar, O. Waddell, T. E. Brooks, B. W. A double antibody sandwich enzyme-linked immunosorbent assay for the detection of Salmonella using biotinylated monoclonal antibodies. J. Food Prot. 2001, 64,1166-1171. 

Enzyme-linked sandwich assay using magnetic particle-labelled probe hybridising to a biotinylated DNA target that captures streptavidin-AP... 

The important function of biotin is its role as coenzyme for carboxylase, which catalyses carbon dioxide fixation or carboxylation reaction. The epsilon amino group of lysine in carboxylase enzymes combines with the carboxyl group of biotin to form covalently linked biotinyl carboxyl carrier protein (BCCP or biocytin) (Figure 6.8). This serves as an intermediate carrier of carbon dioxide. The carboxylation of acetyl CoA to malonyl CoA in presence of acetyl CoA carboxylase requires biotin as coenzyme. Propionyl carboxylase and pyruvate carboxylase are also associated with biotin. 

For the determination of binding properties of biotinylated proteins and conjugates, use flexible 96-wells microplates (Nunc). The general design of the method is similar to classical enzyme-linked immunosorbent assay (ELISA) and RIA methods. Use the described procedure for immobilization of streptavidin, antigen, biotinylated or nonmodified antibody, or catalase. 

A companion approach is to form copolymers using pyrrole and pyrrole butyric acid. The copolymer presents available carboxylic acid groups at the surface that may serve to covalently immobilize the IgG. Figure 6 illustrates the covalent tethering of amine-functionalized biotin following EDC/sulfo-NHS activation of the pendant carboxylic acid groups of poly(pyrrole-co-pyrrolylbutyric acid). Subsequent incubation in streptavidin or neutravidin followed by incubation in biotinylated IgG immobilizes the primary antibody to the device surface. This format has been used to build sandwich immunoassays for antigens that are detected by the indirect action of the oxidoreductase enzyme-linked antibodies on the conductivity of the polymer film. 

It is possible to perform an enzyme-linked immunosorbent assay (ELISA) using a streptavidin-coated 96-well plate the primary antibody, biotinylated-rabbit-anti-streptavidin, specifically recognizes the avidin linked to the helicate as the bioprobe. The detection limit is better than that with commercially available organic dyes such as antistreptavidin-FICT (FiB3). 

Fig. 2. Diagram illustrating the molecular interactions of the LAB procedure. Horseradish peroxidase is covalently linked to avidin. The primary antibody against the antigen of interest is linked to the enzyme labeled avidin complex (LAB) via a biotinylated secondary antibody raised against immunoglobulin of the animal species used to generate the primary antibody (A, immunoglobulin CCC, long carbon arm extension , biotin A, avidin , peroxidase).

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