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- Title
Photometric and Electrochemical Enzyme-Multiplied Assay Techniques Using β-Galactosidase as Reporter Enzyme.
- Authors
Francis H. Ko; Harold G. Monbouquette
- Abstract
β-Galactosidase (β-gal) is shown to be a versatile new reporter enzyme in both photometric and electrochemical enzyme-multiplied assay techniques (EMATs). The well-known β-gal substrate analog, o-nitrophenyl β-d-galactopyranoside, yields the visibly colored, o-nitrophenol product upon hydrolysis, whereas the substrate, p-aminophenyl β-d-galactopyranoside, gives rise to an electrooxidizable product, p-aminophenol. These β-gal substrates made possible the demonstration of both photometric and electrochemical signal transduction schemes for β-gal-based EMAT detection of estradiol (as the estradiol-bovine serum albumin (E-BSA) conjugate). The EMAT system is composed of the reporter enzyme, β-gal, with covalently attached estradiol, and estrogen antibody, which inhibits enzyme activity of the β-gal-estradiol conjugate up to ∼75%. Reporter enzyme inhibition is relieved significantly by addition of ≤2 ng/mL of estradiol (as E-BSA), which competes for binding with the antibody. Thus, the presence of analyte (E-BSA) is reported by the enzyme (β-gal), which amplifies the ligand-protein dissociation event by turning over its substrate repeatedly. The electrochemical version of EMAT, based on amperometric detection of p-aminophenol, is responsive to added estradiol within minutes. These results show that β-gal may serve as a useful alternative to glucose-6-phosphate dehydrogenase, which currently is used as reporter enzyme in commercially available EMAT systems.
- Subjects
ENZYMES; BLOOD plasma; MICROBIAL genetics; BLOOD proteins
- Publication
Biotechnology Progress, 2006, Vol 22, Issue 3, p860
- ISSN
8756-7938
- Publication type
Article