Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids.
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.
Boguslawski
S J
SJ
Smith
D E
DE
Michalak
M A
MA
Mickelson
K E
KE
Yehle
C O
CO
Patterson
W L
WL
Carrico
R J
RJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D004247:DNA / Q000276:immunology*; D004275:DNA, Ribosomal / Q000276:immunology; D004926:Escherichia coli; D007118:Immunoassay / Q000379:methods; D007163:Immunosorbent Techniques; D051379:Mice; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000276:immunology*; D012335:RNA, Ribosomal / Q000276:immunology
D000911:Antibodies, Monoclonal; D004275:DNA, Ribosomal; D012335:RNA, Ribosomal; D012313:RNA; D004247:DNA