Interaction of immobilized avidin with an aequorin - Biotin conjugate: An aequorin-linked assay for biotin

Agatha Feltus, Sridhar Ramanathan, Sylvia Daunert

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

Biotinylated recombinant aequorin was used in the development of a heterogeneous bioluminescence binding assay for biotin. This assay is based on a competition between a biotinylated aequorin conjugate and biotin for the binding sites of avidin immobilized on solid particles. Dose-response curves were obtained that relate solid-phase aequorin activity to the concentration of biotin. Under certain experimental conditions these curves were biphasic; i.e., as the biotin concentration increased, the solid-phase aequorin activity first increased reaching a maximum and then decreased at higher biotin concentrations. This 'hook' effect was observed with four different types of immobilization supports. The effect was more pronounced when low concentrations of aequorin-biotin conjugate were used, and diminished at a high conjugate concentration. This behavior indicates a possible positive cooperativity in the interaction between the immobilized avidin and biotin. Scatchard plot analysis was also consistent with a positive cooperativity mechanism. By using the ascending portion of the dose-response curve, the detection limit of the assay for biotin was 1 x 10-15 M (100 zmol of biotin in the sample).

Original languageEnglish (US)
Pages (from-to)62-68
Number of pages7
JournalAnalytical Biochemistry
Volume254
Issue number1
DOIs
StatePublished - Dec 1 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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