Sensitive radioimmuno assay for 2',5'-oligoadenylates using a novel 125I-labeled derivative of 2',5'-triadenylate 5'-triphosphate.
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 periodate-oxidized pA2'p5'A2'p5'A with beta-alanyltyrosine 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'-oligoadenylates 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 microliter 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.
D015088:2',5'-Oligoadenylate Synthetase / Q000378:metabolism; D000227:Adenine Nucleotides / Q000032:analysis* / Q000276:immunology; D000818:Animals; D000918:Antibody Specificity; D002478:Cells, Cultured; D003429:Cross Reactions; D007370:Interferon Type I / Q000494:pharmacology; D009843:Oligoribonucleotides / Q000031:analogs & derivatives / Q000032:analysis* / Q000276:immunology; D011863:Radioimmunoassay
D000227:Adenine Nucleotides; D007370:Interferon Type I; D009843:Oligoribonucleotides; C023505:2',5'-oligoadenylate; D015088:2',5'-Oligoadenylate Synthetase