Photometric and electrochemical enzyme-multiplied assay techniques using beta-galactosidase as reporter enzyme.
Beta-galactosidase (beta-gal) is shown to be a versatile new reporter enzyme in both photometric and electrochemical enzyme-multiplied assay techniques (EMATs). The well-known beta-gal substrate analog, o-nitrophenyl beta-d-galactopyranoside, yields the visibly colored, o-nitrophenol product upon hydrolysis, whereas the substrate, p-aminophenyl beta-D-galactopyranoside, gives rise to an electrooxidizable product, p-aminophenol. These beta-gal substrates made possible the demonstration of both photometric and electrochemical signal transduction schemes for beta-gal-based EMAT detection of estradiol (as the estradiol-bovine serum albumin (E-BSA) conjugate). The EMAT system is composed of the reporter enzyme, beta-gal, with covalently attached estradiol, and estrogen antibody, which inhibits enzyme activity of the beta-gal-estradiol conjugate up to approximately 75%. Reporter enzyme inhibition is relieved significantly by addition of < or =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 (beta-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 beta-gal may serve as a useful alternative to glucose-6-phosphate dehydrogenase, which currently is used as reporter enzyme in commercially available EMAT systems.
Ko
Francis H
FH
Biomedical Engineering Interdepartmental Graduate Program, University of California, Los Angeles, Los Angeles, California 90095, USA.
Monbouquette
Harold G
HG
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D004563:Electrochemistry; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D017248:Enzyme Multiplied Immunoassay Technique; D004926:Escherichia coli / Q000201:enzymology; D004958:Estradiol / Q000032:analysis / Q000378:metabolism; D008024:Ligands; D010783:Photometry; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D000906:Antibodies; D004791:Enzyme Inhibitors; D008024:Ligands; D012710:Serum Albumin, Bovine; D004958:Estradiol; D001616:beta-Galactosidase