Interaction of Immobilized Avidin with an Aequorin–Biotin Conjugate: An Aequorin-Linked Assay for Biotin

✍ Scribed by Agatha Feltus; Sridhar Ramanathan; Sylvia Daunert

• Publisher: Elsevier Science
• Year: 1997
• Tongue: English
• Weight: 215 KB
• Volume: 254
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1997.2400

✦ Synopsis

to labeled biotin results in an inhibition of the signal Biotinylated recombinant aequorin was used in the generated by the label (3-8). The higher the amount development of a heterogeneous bioluminescence of free biotin present, the less labeled biotin is bound binding assay for biotin. This assay is based on a comto avidin, and the higher the signal observed. In a hetpetition between a biotinylated aequorin conjugate erogeneous competitive binding system, the labeled biand biotin for the binding sites of avidin immobilized otin competes with biotin in solution for avidin immobion solid particles. Dose-response curves were oblized on a solid phase (9-13). Separation of the phases tained that relate solid-phase aequorin activity to the removes unbound labeled biotin. The signal generated concentration of biotin. Under certain experimental by the labeled biotin bound to avidin is then measured, conditions these curves were biphasic; i.e., as the bioand the signal can be related to the concentration of tin concentration increased, the solid-phase aequorin biotin present in the sample.

activity first increased reaching a maximum and then

In general, binding assays produce a dose-response decreased at higher biotin concentrations. This ''hook'' curve which is sigmoidal in shape, reaching a plateau effect was observed with four different types of immoat high concentrations of ligand. Under certain circumbilization supports. The effect was more pronounced stances, however, biphasic curves in which the dosewhen low concentrations of aequorin-biotin conjuresponse curve has a maximum at low concentrations gate were used, and diminished at a high conjugate of ligand have been observed (14-18). These ''hook'' concentration. This behavior indicates a possible posieffects have been theorized to be caused by either the tive cooperativity in the interaction between the imformation of a cyclic complex (19-22) or positive coopmobilized avidin and biotin. Scatchard plot analysis erativity between ligand binding sites on the same was also consistent with a positive cooperativity mechbinder (23-27).

anism. By using the ascending portion of the doseresponse curve, the detection limit of the assay for

The photoprotein aequorin has been recently exbiotin was 1 1 10 015 M (100 zmol of biotin in the samplored as a label in the development of bioluminescence ple). ᭧ 1997 Academic Press assays (7,(28)(29)(30)(31)(32)(33). In this study, an aequorin-biotin conjugate along with avidin-coated particles was used to develop a heterogeneous bioluminescence binding assay for biotin. The dose-response curves obtained Biotin and its binding protein avidin have been used were biphasic in nature with a hook effect at low conextensively in the development of a variety of bioanacentrations of biotin. By using the ascending portion of lytical techniques that take advantage of the extremely the curve, a detection limit of 1 1 10 015 M biotin was high affinity (K d É 10 015 M) between the two biomoleobtained. Because of the possibility that these hook cules (1, 2). This has been especially effective in the effects can be caused by positive cooperative binding, development of homogeneous and heterogeneous comthe nature of the interaction between immobilized avipetitive binding assays in which biotin in the sample din and biotinylated aequorin was also studied by competes with labeled biotin for the binding sites of Scatchard plot analysis. Given the low value of the K d , avidin. In a homogeneous system, the binding of avidin it is the inherent sensitivity afforded by the aequorin label that enables the performance of Scatchard analysis in this system. Several groups have previously in-