An improved preparation and purification of oligonucleotide-alkaline phosphatase conjugates

A simple, low-cost protocol giving good yields of oligonucleotide-alkaline phosphatase conjugates on a 7-nmol or a 35-nmol scale of oligomer has been developed. The cross-linking agent is disuccinimidyl suberate. n-Butanol is used to remove excess disuccinimidyl suberate and side products away from the disuccinimidyl suberate/oligomer adduct before alkaline phosphatase is added directly to the dried adduct. The crude conjugate is purified in one step using a DEAE HPLC column and an NaCl gradient. These conjugates were used to detect 0.4 pg of a hepatitis B virus sequence using a chemiluminescent assay.