Comparison of an Intercalating Dye and an Intercalant−Enzyme Conjugate for DNA Detection in a Microtiter-Based Assay

Abstract

Two methods have been developed for the detection of DNA immobilized on the surface of microtiter wells. An intercalating dye, 3,6-diaminoacridine, is used in stain and rinse solutions, so that measured absorbance values (450 nm) reflect the sum of DNA-bound and free dye. With diaminoacridine, signal increases of 0.056 ± 0.010 were achieved on immobilizing double-stranded calf thymus DNA. An intercalant−enzyme conjugate, consisting of an average of four daunomycin moieties covalently bound to each glucose oxidase, was shown to provide a 10-fold signal enhancement (optimum 0.25 μM, with rinsing and peroxidase-o-dianisidine detection) compared to diaminoacridine, due to catalytic amplification; signals of 0.50 ± 0.05 were obtained. This conjugate possesses 56% of the activity of native glucose oxidase and was prepared using water-soluble carbodiimide and N-hydroxysuccinimide reagents. Single-stranded DNA was immobilized onto avidin-coated polystyrene plates and commercially available (Covalink) plates possessing secondary amine groups. Following hybridization with complementary DNA, detection was performed with the daunomycin−glucose oxidase conjugate. Both immobilization methods showed optimum DNA concentrations of 0.10 μg/mL, and maximum signal intensities were obtained when >0.5 μg/mL complementary DNA was present in the hybridization solution. Some nonspecific binding of the intercalant−enzyme conjugate was suggested by results obtained with avidin-coated polystyrene plates, but not with Covalink plates.