Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids

• Published in: Journal of immunological methods Vol. 89; no. 1; p. 123
• Main Authors: Boguslawski, S J, Smith, D E, Michalak, M A, Mickelson, K E, Yehle, C O, Patterson, W L, Carrico, R J
• Format: Journal Article
• Language: English
• Published: Netherlands 01.05.1986
• Subjects: Animals; Antibodies, Monoclonal - immunology; Antibody Specificity; DNA - immunology; DNA, Ribosomal - immunology; Escherichia coli; Immunoassay - methods; Immunosorbent Techniques; Mice; Nucleic Acid Hybridization; RNA - immunology; RNA, Ribosomal - immunology
• ISSN: 0022-1759
• DOI: 10.1016/0022-1759(86)90040-2

Monoclonal antibodies were raised to a DNA.RNA heteropolymer duplex prepared by transcription of phi X174 single-stranded DNA with DNA-dependent RNA polymerase. A monoclonal antibody with the highest affinity and specificity was selected. This antibody bound the DNA.RNA heteropolymer and poly(I).poly(dC) equally but 100-fold higher levels of poly(A).poly(dT) were required to achieve a similar degree of binding in competitive binding assays using DNA.[3H]RNA. Single-stranded DNA, double-stranded DNA and RNA, and ribosomal RNA were not bound by the antibody. The observed association constant for the antibody and DNA.[3H]RNA, determined by Scatchard analysis, was 8.5 X 10(10) l/mol assuming independent antibody binding sites. The antibody and an alkaline phosphatase-labeled second antibody were used in an immunodetection method for measurement of hybrids formed between immobilized DNA probes of various lengths and 23 S ribosomal RNA. The colorimetric response of this assay increased linearly with the amount of hybrid formed.