Sensitive Radioimmuno Assay for 2′,5′-Oligoadenylates Using a Novel 125I-Labeled Derivative of 2′,5′-TriadenyIate 5′-Triphosphate

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Abstract
A novel 125I-labeled derivative of 2′,5′-triadenylate 5′-triphosphate, pppA2′p5′A2′-p5′A, with high specific radioactivity was synthesized by coupling of periodateoxidized pA2′p5′A2′p5′A with β-alanyItyrosine methyl ester followed by 5′-triphosphorylation and iodination with 125I. Antisera toward 2′,5′-oligoadenylate 5′-triphosphate were produced in rabbits by immunization with the conjugate of pppA2′p5′A2′p5′A2′p5′A with bovine serum albumin, and an antiserum with high specificity and high sensitivity for 2′,5′-oligoadenylates was selected and tested extensively. Radioimmuno assaying of 2′,5′-oIigoadenylates was carried out by a competitive double antibody method in which the amount of the antibody bound to the 125I-labeled probe was measured after precipitation with goat anti-rabbit IgG. The concentration of pppA2′p5′A2′p5′A required for 50% inhibition of the binding between the antiserum and the probe was 0.6 nM. The cross reactivity of the antiserum with the 3′,5′-triadenylate was more than 10,000 times weaker compared to in the case of 2′,5′-oligoadenylates. Very low or no cross reaction was observed with ATP, AMP, and adenosine. The radioimmuno assay using the 125I-labeled compound and the antiserum allows the direct analysis of 2′,5′-oligoadenylates in the range of 4 fmol to 1 pmol (0.04–10 nM in a 100 μl sample). This assay was applied to the measurement of the activity of 2′,5′-oligoadenylate synthetase in cells stimulated by interferon. The properties of the 125I-labeled derivative of pppA2′p5′A2′p5′A are described.

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