A radioimmunoassay for guanosine-5'-diphosphate-3'-diphosphate and adenosine-5'-triphosphate-3'-diphosphate

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'-triphosphate-3'-diphosphate with human serum albumin using glutaraldehyde, respectively. Antibody-bound 3H-labeled highly phosphorylated nucleotides were separated from the free 3H-labeled 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 convenience compared with the previous 32PO4-labeling technique.