Homogeneous enzyme immunoassay modified for application to luminescence-based biosensors

Application of immunoassay to biosensors for use in the point-of-care setting ideally requires immunoassay without separation steps and with small volumes of both sample and reagents. The suitability of cloned enzyme donor immunoassay (CEDIA), one of a few homogeneous immunoassays available, was investigated for application to biosensors. This method is based on the bacterial enzyme β-galactosidase, which has been genetically engineered by others into two inactive fragments, enzyme donor (ED) and enzyme acceptor (EA). Association of the ED and EA fragments in the assay results in formation of active enzyme, which acts on substrate to generate a detectable signal. Sensitivity of commercially available CEDIA kits were compared, with respect to the sample and reagent volumes, using three different signal generation processes. The CEDIA kit for valproic acid and three substrates, a colorimetric (chlorophenol red-β-d-galactopyranoside), a chemiluminescent (Lumi-Gal 530), and a bioluminescent (Beta-Glo Assay System), were employed in the study. Our results indicate that the high sensitivity of the bioluminogenic substrate, d-luciferin-O-β-galactopyranoside, with short assay time and small volumes of sample and reagents required for the assay, simple handling, and relatively low expense, make this substrate, together with CEDIA, suitable for application to biosensors intended for drug and metabolite monitoring in the point-of-care setting.