抗DNA.RNA单克隆抗体的特性及其在杂交体免疫检测中的应用。

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

作者信息

Boguslawski S J, Smith D E, Michalak M A, Mickelson K E, Yehle C O, Patterson W L, Carrico R J

出版信息

J Immunol Methods. 1986 May 1;89(1):123-30. doi: 10.1016/0022-1759(86)90040-2.

Abstract

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.

摘要

用依赖于DNA的RNA聚合酶转录φX174单链DNA制备的DNA.RNA杂聚物双链体产生了单克隆抗体。选择了具有最高亲和力和特异性的单克隆抗体。该抗体与DNA.RNA杂聚物和聚（I）.聚（dC）的结合程度相同，但在使用DNA.[3H]RNA的竞争性结合试验中，需要100倍更高水平的聚（A）.聚（dT）才能达到类似的结合程度。单链DNA、双链DNA和RNA以及核糖体RNA均不与该抗体结合。通过Scatchard分析确定，假设抗体结合位点独立，该抗体与DNA.[3H]RNA的观察到的缔合常数为8.5×10(10) l/mol。该抗体和碱性磷酸酶标记的第二抗体用于免疫检测方法，以测量各种长度的固定化DNA探针与23 S核糖体RNA之间形成的杂交体。该测定的比色响应随形成的杂交体数量呈线性增加。