Development of an enzyme-linked immunosorbent assay incorporating the enzyme alcohol oxidase and the reporter peroxyfluor-1

CHED 952

Katherine A Leehy, kaleehy@stthomas.edu, Laura J Baumgartner, and Katherine Olson. Department of Chemistry, University of Saint Thomas, 2115 Summit Ave, Saint Paul, MN 55105
Alcohol oxidase (AOX) and Peroxyfluor-1 (PF1) is utilized to make a sensitive enzyme-linked immunosorbent assay (ELISA) that can be used to test the concentration of biological species in solution. AOX is an enzyme from Pichia pastoris that has the ability to convert alcohols into hydrogen peroxide. Hydrogen peroxide then converts the PF1 into fluorescein, a highly fluorescent chemical. A competition ELISA can be set up with an analyte, such as estradiol, from the unquantified source and a known amount of the analyte linked to AOX. The change in fluorescence is then quantified to determine the concentration of the analyte in the unknown sample. A low quantifiable change in fluorescence would correlate with a high concentration in the sample because the analyte in the sample would outcompete the AOX-analyte conjugate. Quantifying these unknown samples can be used to test for varied health conditions associated with changes in hormone levels.