THE PRECISE RADIOIMMUNOASSAY OF ADENOSINE - MINIMIZATION OF SAMPLE COLLECTION ARTIFACTS AND IMMUNOCROSSREACTIVITY

Anti-adenosine antibodies were produced in rabbits immunized with N6-carboxymethyladenosine conjugated to methyl albumin. 125I-N6-Aminobenzyladenosine was synthesized and used as a high-specific-activity, high-affinity ligand. A radioimmunoassay (RIA) was developed that can detect 6.25 nm (312.5 fmol) of underivatized adenosine and cross-reacts < 0.02% with adenine nucleotides and guanosine and not at all with 1 mm inosine. The sensitivity of the RIA can be increased to a detection limit of 0.125 nm (6.25 fmol) by derivitizing samples with benzyl bromide to form N6-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 ZnSO4 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. © 1992.