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 sensitivity of an enzyme-linked oligosorbent assay diagnostic test. To understand the influence of their structure and conformation in solution on the efficiency of the test during the capture step, two different ways of synthesizing these conjugates were compared. The first consisted of coupling 5' amino modified oligonucleotides to poly(maleic anhydride-alt-methylvinyl ether) and poly(maleic anhydride-alt-ethylene). The second resulted from direct synthesis of oligonucleotides from poly(maleic anhydride-alt-ethylene) previously grafted onto a controlled pore glass support. The different conjugates 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 bioMérieux VIDAS instrument. Different parameters were studied, such as the purity of the conjugate solution 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 demonstrated that conjugates produced an amplification factor of 50 versus the capture oligonucleotide probe used as the reference. The detection limit reached 10(8) copies/mL.
Minard-Basquin
C
C
UMR 2142 CNRS/bioMérieux, ENS Lyon, 46 allée d'Italie, 69364 Lyon Cedex 7, France.
Chaix
C
C
Pichot
C
C
Mandrand
B
B
D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D011933:Reagent Kits, Diagnostic / Q000592:standards*
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D011108:Polymers; D011933:Reagent Kits, Diagnostic