HOMOGENEOUS BIOLUMINESCENCE COMPETITIVE-BINDING ASSAY FOR FOLATE BASED ON A COUPLED GLUCOSE-6-PHOSPHATE DEHYDROGENASE-BACTERIAL LUCIFERASE ENZYME-SYSTEM

A homogeneous bioluminescence competitive binding assay for folate was developed by using a coupled enzyme system of glucose-6-phosphate deh ydrogenase (G6PDH) and bacterial luciferase. A highly substituted G6PD H-folate conjugate was prepared by employing an N-hydroxysuccinimide/c arbodiimide method. Folate binding protein inhibits the activity of th e conjugate. In the presence of folate, there is a competition between folate and the G6PDH-folate conjugate for the binding site of the fol ate binding protein, and the activity of the conjugate is recovered. T hus, the concentration of folate can be related to the activity of the G6PDH-folate conjugate, which is directly related to the bioluminesce nce produced by the coupled enzyme reaction. Using this assay, dose-re sponse curves with a detection limit of 2.5 x 10(-8) M folate were obt ained, which is an improvement of an order of magnitude with respect t o an assay that monitors G6PDH activity spectrophotometrically. The as say was validated using vitamin tablets and a cell culture medium.