Enzyme-Linked Competitive Binding Assays Based on the Biotin/Avidin Interaction

Abstract

The strong interaction between biotin and avidin is utilized for the development of enzyme-linked competitive binding assays not only for biotin itself, but also for other biomolecules. Alkaline phosphatase, a single substrate enzyme typically used for heterogeneous types of competitive binding assays, is employed also for homogeneous types. For the biotin assay, the heterogeneous protocol offers a much improved detection capability when compared to the homogeneous type. A simple approach of simulating dose-response curves is introduced. An effective analyte concentration attached to an enzyme label is determined by using the same binder, but with a different enzyme label. The analyte system adapted to the biotin/avidin-mediated homogeneous protocol is digoxin and a monoclonal anti-digoxin antibody. The detection capabilities of these assays, particularly the homogeneous types, are shown to be limited by the detectability of the enzyme conjugate employed.

Template Fabrication of Protein-Functionalized Gold−Polypyrrole−Gold Segmented Nanowires

“…(2) Total Biotin Binding Sites. A modification of an indirect enzyme-linked binding assay method 20 was employed to estimate the total number of biotin binding sites per nanowire. The method is based on the fact that the reaction between an enzyme-biotin conjugate and avidin reduces the catalytic activity of the enzyme by altering its tertiary structure and/ or because of steric effects.…”

A magnetoelastic bioaffinity-based sensor for avidin

“…The interaction between avidin (or streptavindin) and biotin exhibits the strongest known affinity (dissociation constant, K d ∼ 10 15 M −1 ) between a ligand and a protein (Weber et al, 1989). The high specificity of avidin for biotin has resulted in the avidin-biotin system being extensively used in biological science and technology, including affinity-based separation, diagnostic assays and DNA hybridization (Cho et al, 1999;Masarik et al, 2003). Avidin is toxic to many organisms due to its ability to deplete biotin, an essential vitamin (Vitamin H), from their environment (Mock et al, 2003).…”

Development of Bioluminescence Immunoassay Using Photoprotein, Aequorin and Site-directed Immobilization

“…As a signal generator, bioluminescent reporter proteins have been used as an excellent label, which have advantages of low background, large dynamic range, high sensitivity, and no need of substrate or enhancer in mmunoassay systems. Several immunoassays have been developed for determining digoxin using enzymes and mutant aequorin as a label, [13][14][15] however, it is the first time to develop the bioluminescence immunoassay by employing native aequorin for digoxin assay.…”

“…The resulting aequorin conjugates were characterized by their residual activities. [13][14][15][16] All conjugates were kept at 4 °C until the additions of reagents for activity measurements.…”