MONOCLONAL-ANTIBODIES TARGETED TO ALPHA-OLIGONUCLEOTIDES - CHARACTERIZATION AND APPLICATION IN NUCLEIC-ACID DETECTION

The aim of the present study was to test the antigenicity of alpha-deo xyribonucleotides 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 alpha-deoxyribonucl eotides. Two were raised against a poly(alpha-dT) sequence and specifi cally recognized the alpha-dT nucleotide. Two were raised against a se quence containing all four common nucleotides as alpha-nucleotides and , surprisingly, only recognized the alpha-dG nucleotide. For all four Mabs, no cross reactivity was observed with beta-oligonucleotides. The se Mabs were reactive with alpha-oligonucleotide sequences whether the se sequences were single-stranded or hybridized to DNA or RNA, The fou r Mabs were tested in a sandwich hybridization assay that consisted of an alpha-oligonucleotide (for target sequence recognition), one of th e four Mabs (far recognition of the hybridized alpha-oligonucleotide), and goat anti-mouse antibody conjugated to horse radish peroxydase (H RP) (for detection). One of the monoclonal antibodies, Mab 2E11D7, was directly conjugated to HRP and used in sandwich hybridization to dete ct 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.