An Avidin-Biotin Solid Phase ELISA for Femtomole Isopentenyladenine and Isopentenyladenosine Measurements in HPLC Purified Plant Extracts

Abstract

A solid phase enzyme immunoassay was developed for isopentenyladenine (iP) and isopentenyladenosine (iPA) quantitation in HPLC purified plant extracts. It was performed on antigen-coated microtitration plates on which bound antibodies were indirectly labeled by the means of a biotinylated goat anti-rabbit antibody and an avidin-alkaline phosphatase conjugate. Less than 3 femtomoles of iP or iPA were easily detected and the measuring range extended from 3 femtomole to 1 picomole. The reproducibility has been tested and intra- and interassay variations did not exceed 5.0%. The specificity of iPA antibodies was good, as determined by cross-reactivity measurements with other adenylic compounds. The specificity of the measurements for iP and iPA was demonstrated by analysis of the immunoreactivity of fractions obtained by HPLC separation of a methanolic tobacco leaf extract.