Oligonucleotide-polymer conjugates: Effect of the method of synthesis on their structure and performance in diagnostic assays

Oligonucleotide-polymer conjugates have been described to improve the sensi tivity of an enzyme-linked oligosorbent assay diagnostic test. To understan d the influence of their structure and conformation in solution on the effi ciency of the test during the capture step, two different ways of synthesiz ing these conjugates were compared. The first consisted of coupling 5' amin o modified oligonucleotides to poly(maleic anhydride-alt-methylvinyl ether) and poly(maleic anhydride-alt-ethylene). The second resulted from direct s ynthesis of oligonucleotides from poly(maleic anhydride-alt-ethylene) previ ously grafted onto a controlled pore glass support. The different conjugate s were analyzed by size-exclusion chromatography and viscometry. The former method for conjugate synthesis produced aggregates, which was not the case for the latter. These conjugates were then used in the capture phase of a hybridization assay using a HBV DNA target, on a bioMerieux VIDAS instrumen t. Different parameters were studied, such as the purity of the conjugate s olution and the number of oligonucleotides per polymer chain. The amount of conjugate coated on the solid-phase receptacle surface at the time of the capture phase was evaluated by radioactive labeling. Finally, it was demons trated that conjugates produced an amplification factor of 50 versus the ca pture oligonucleotide probe used as the reference. The detection limit reac hed 10(8) copies/mL.