Monoclonal antibodies targeted to a-oligonucleotides. Characterisation and application in nucleic acid detection

Abstract

The aim of the present study was to test the antigenicity of α-deoxyribonucleotides in order to develop a new tool for the detection of nucleic acid sequences for use in diagnostic applications. We describe four monoclonal antibodies (Mabs) which recognize α-deoxyribonucleotides. Two were raised against a poly(α-dT) sequence and specifically recognized the α-dT nucleotide. Two were raised against a sequence containing all four common nucleotides as α-nucleotides and, surprisingly, only recognized the α-dG nucleotide. For all four Mabs, no cross reactivity was observed with /3-oligonucleotides. These Mabs were reactive with aoligonucleotide sequences whether these sequences were single-stranded or hybridized to DNA or RNA. The four Mabs were tested in a sandwich hybridization assay that consisted of an α-oligonucleotide (for target sequence recognition), one of the four Mabs (for recognition of the hybridized a-oligonucleotide), and goat anti-mouse antibody conjugated to horse radish peroxydase (HRP) (for detection). One of the monoclonal antibodies, Mab 2E11D7, was directly conjugated to HRP and used in sandwich hybridization to detect PCR fragments of HPV 18 DNA. The sensitivity of this reaction was 1 pg of plasmid DNA containing the HPV 18 fragment. The specificity of the detection was demonstrated using HPV 6/11 and 16 DNA sequences. © 1994 Oxford University Press.