The precise radioimmunoassay of adenosine : minimization of sample collection artifacts and immunocrossreactivity

Abstract Anti-adenosine antibodies were produced in rabbits immunized with N 6 -carboxymethyladenosine conjugated to methyl albumin. 125 I- N 6 -Aminobenzyladenosine was synthesized and used as a high-specific-activity, high-affinity ligand. A radioimmunoassay (RIA) was developed that can detect 6.25 n m (312.5 fmol) of underivatized adenosine and cross-reacts m inosine. The sensitivity of the RIA can be increased to a detection limit of 0.125 n m (6.25 fmol) by derivitizing samples with benzyl bromide to form N 6 -benyladenosine. The assay was adapted to an automated RIA procedure. Assay precision was increased by: (i) inhibiting slight adenosine deaminase activity present in antisera; (ii) treating buffers and albumin used in the RIA with charcoal to remove contaminating adenosine; and (iii) correcting for a small but variable component of immunoreactivity not attributable to adenosine. A second antibody prepared with a 2′,3′-disuccinyladenosine-albumin conjugate was also found to detect some non-adenosine-mediated immunoreactivity in plasma samples. Immunointerference in human plasma was eliminated in samples treated with ZnSO 4 Ba(OH) 2 or partially purified over C18 Sep Paks to remove nucleotides and assayed after sample benzylation or succinylation. Human blood was mixed with a novel “stop” solution that was optimized to inhibit adenosine formation from AMP by > 99% and to inhibit adenosine uptake into red cells and degradation by > 94%. Human plasma/stop solution was assayed by RIA and HPLC with equivalent results.