Potentiometric Homogeneous Enzyme-Linked Competitive Binding Assays Using Adenosine Deaminase as the Label

Thea L. Kjellström, Leonidas G. Bachas

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

Homogeneous enzyme-linked competitive binding assays for biotin are described that are based on the competition between an enzyme-biotin conjugate and free biotin for a fixed number of binding sites of avidin. Unlike conventional homogeneous enzyme immunoassays, in this system the analyte (biotin) is labeled with adenosine deaminase (ADA), an ammonia-producing enzyme. Consequently, potentiometric rather than photometric methods can be used as means of detection. Several ADA-blotin conjugates were prepared and showed as high as 97% inhibition of the enzymatic activity in the presence of avidin. Addition of free biotin reverses this inhibition in an amount proportional to the concentration of analyte. Relatively steep dose-response curves were observed, leading to a precise and accurate assay for biotin. The detection limits of these curves were as low as 1 × 10−8 M. Varying the concentration of the reagents in the assay allowed the detection limit and working range to be altered to a desired value. The proposed method was applied in the determination of biotin in a horse-feed supplement.

Original languageEnglish (US)
Pages (from-to)1728-1732
Number of pages5
JournalAnalytical Chemistry
Volume61
Issue number15
DOIs
StatePublished - Aug 1989
Externally publishedYes

ASJC Scopus subject areas

  • Analytical Chemistry

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