Monoclonal antibodies to 5'-triphospho-(2'-5')adenyladenosine oligonucleotides.

Abstract

Thirteen monoclonal antibodies to ppp(A2'p5')nA oligonucleotides have been produced by fusing Y3 myeloma cells with spleen cells of rat hyperimmunized with A2'p5'A succinyl albumin as immunogen. 125I-labeled A2'p5'A succinyl tyrosine methyl ester was used as a labeled probe. Antibodies were detected by their ability to bind the labeled analog and were selected for their affinity for A2'p5'A. There were no significant differences between the properties of the monoclonal antibodies and of the antiserum. They discriminated between 2'-5' and 3'-5' phosphodiester bonds: they crossreacted poorly with (A3'p5')2A (crossreactivity ratio, greater than 10(4)) and even less with ATP an adenosine (crossreactivity ratio, greater than (6)). The affinity was high (Kd = 6 x 10(-12) M) for succinyl A2'p5'A, which is the best ligand, and also high for A2'p5'A (crossreactivity ratio, 3) and (A2'p5')2A, (A2'p5')3A, and (As'p5')4A (crossreactivity ratio, 7.5). The binding to triphosphorylated isomers, ppp(A2'p5)nA, was affected by the presence of the triphosphate groups, and the affinity increased as the length of the isomer increased (crossreactivity ratio, 10,000 for n = 1 to 200 for n = 4). Thermodynamic analysis of these data demonstrated that, in dephosphorylated isomers, the binding site of highest affinity was located at the 5'-OH end of the molecule whereas in phosphorylated isomers, the favorable binding sites were in the middle and at the 2'-OH end of the chain. Use of monoclonal antibodies of such a specificity together with the 125I-labeled 2-5A analog allows quantification of (A2'p5')nA directly and of ppp(A2'p5')nA after removal of the terminal phosphates by alkaline phosphatase treatment.

Immunological evidence for the in vivo occurrence of (2'-5')adenylyladenosine oligonucleotides in eukaryotes and prokaryotes.

“…The monoclonal antibody and 125I-labeled A2'p5'A-succinyltyrosine methyl ester were produced in our laboratory as described (24,25 (24,25). Briefly, frozen tissues were ground in 9 vol of cold 1 M perchloric acid.…”

“…We have recently developed a radioimmunoassay using a monoclonal antibody that displays a high affinity for the (2'-5')adenylyladenosine, the recurring unit of (2'-5')An oligomers (24,25). It allows the measurement of nonphosphorylated oligomers at once and of phosphorylated oligomers after removal of the 5'-phosphates.…”

“…Aliquots of the neutral extracts were brought to suitable dilutions with distilled water prior to being assayed. To other aliquots were added the appropriate buffer and enzyme solutions (25). Enzymatic reactions were stopped by heating at 90'C.…”

“…Enzymatic reactions were stopped by heating at 90'C. The samples were then diluted 1:4 to avoid salt interferences in the radioimmunoassay (25). Binding measurements were performed as described (24); details are given in the legend of Table 1.…”

The 2–5 A system: Modulation of viral and cellular processes through acceleration of RNA degradation

“…Radioimmunoassay. The minimal structure recognized is -A2'pA2'pA- (Knight et al, 1981), -AZ'pA- (Cailla et al, 1982), or (pp)pAZ'pA- (Sawai et al, 1985a). B.…”