Radioimmune and radiobinding assays for A2'p5'A2'p5'A, pppA2'p5'A, and related oligonucleotides.

Abstract

Publisher Summary This chapter describes a simple radioimmune and radiobinding assays that are capable of detecting physiological concentrations (that is, nanomolar) of core and 2-5A, respectively. These assays are based on the high affinities of core for antibody prepared against A2'p5'A2'p5'A bound to bovine serum albumin (BSA) in the radioimmune (RI) assay, and of 2-5A for the 2-5A-dependent RNase in the radiobinding (RB) assay. The major difficulty in the development of these assays lay in the synthesis of radioactive 2-5A of sufficiently high specific activity to give the required sensitivity. The 3' addition of [ 32 P]pCp to 2-5A and core with the T4 RNA ligase provides probes that allow the detection of nanomolar concentrations of 2-5A and core with RB and RI assays, respectively. The RI assay is more sensitive for the nonphosphorylated core whereas the 5'-mono-, di-, and triphosphorylated trimers compete to a decreasing extent in that order. The two assays are, therefore, complementary in their abilities to detect core and 5' phosphorylated 2-5A. The 3'-S'-linked oligoadenylic acids are relatively inactive in both assays as are the individual nucleosides and nucleotides contained in 2-5A and 2-5A-pCp.