A Radioimmunoassay for Guanosine-5′-Diphosphate-3′-Diphosphate and Adenosine-5′-Triphosphate-3′-Diphosphate

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Abstract
A radioimmunoassay for guanosine-5′-.diphosphate-3′-diphosphate (ppGpp) and adenosine-5′-triphosphate-3-diphosphate (pppApp) has been developed. The assay method is based on competition of an unlabeled highly phosphorylated nucleotide with 3H-labeled highly phosphorylated nucleotide for binding sites on a specific antibody. Antibodies to ppGpp and pppApp were obtained by immunizing rabbits with the antigen prepared by conjugating ppGpp with human serum albumin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and with the antigen prepared by conjugating 8-(6-aminohexyl)amino-adenosine-5′-triphos- phate-3′-diphosphate with human serum albumin using glutaraldehyde, respectively. Anti body-bound H-labe1ed highly phosphorylated nucleotides were separated from the free 3 highly phosphorylated nucleotides by selective adsorption on dextran-coated charcoal. Displacement plots were linear over a concentration range of 5–1,000 pmol/assay tube in a log-probit percentage plot. Application of this method to biological systems offers improved accuracy and conven ience compared with the previous 33PO4-labeling technique.

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