TY - JOUR
T1 - Radioimmune and Radiobinding Assays for A2′p5′A2′p5′A, pppA2′p5′A2′p5′A, and Related Oligonucleotides
AU - Knight, M.
AU - Wreschner, D. H.
AU - Silverman, R. H.
AU - Kerr, Ian M.
N1 - Funding Information:
We are indebted to Simon Jones and Colin Reese for the giR of chemically synthesized 2-5A trimer 5'-mono-, di-, and triphosphates and arc very grateful to Bernard Erlanger for advice and the gift of antisera to Y-5'-linked oligoadenylic acids used in initial studies not reported here. M. K. was the recipient of a grant from the government of Ghana, and D. H. W. of an EMBO fellowship. P. J. Cayley and R. E. Brown developed the HPLC methods employed (this volume \[27\]).
PY - 1981/1/1
Y1 - 1981/1/1
N2 - 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 [32P]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.
AB - 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 [32P]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.
UR - http://www.scopus.com/inward/record.url?scp=0019739905&partnerID=8YFLogxK
U2 - 10.1016/S0076-6879(81)79032-3
DO - 10.1016/S0076-6879(81)79032-3
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AN - SCOPUS:0019739905
SN - 0076-6879
VL - 79
SP - 216
EP - 227
JO - Methods in Enzymology
JF - Methods in Enzymology
IS - C
ER -