Combination of DNA-directed immobilization and immuno-PCR: very sensitive antigen detection by means of self-assembled DNA-protein conjugates

An assay for very sensitive antigen detection is described which takes advantage of the self-assembly capabilities of semi-synthetic conjugates of DNA and proteins. The general scheme of this assay is similar to a two-sided (sandwich) enzyme-linked immunoassay (ELISA); however, covalent single-stranded DNA±streptavidin (STV) conjugates, capable of hybridizing to complementary surface-bound DNA oligomers, are utilized for the effective immobilzation of either capture antibodies or anti-gens, rather than the chemi- or physisorption usually applied in ELISA. Immuno-PCR (IPCR) is employed as a method for signal generation, utiliz-ing oligomeric reagents obtained by self-assembly of STV, biotinylated DNA and antibodies. In three different model systems, detecting human IgG, rabbit IgG or carcinoembryonic antigen, this com-bination allowed one to increase the sensitivity of the analogous ELISA ~1000-fold. For example, <0.1 amol/ml (15 pg/ml) of rabbit IgG was detectable. The immunoassay can be carried out in a single step by tagging the analyte with both reagents for capture and read-out simultaneously, thereby sig-ni®cantly reducing handling time and costs of analysis. Moreover, as the spatial selectivity of tar-get immobilization is determined by the speci®city of DNA base pairing, the assay is particularly suited for miniaturized micro¯uidics and lab-on-a-chip devices