Adenosine was coupled to human serum albumin by two different procedures that preserved the purine and ribose rings. The conjugates were evaluated for antigenicity in rabbits and guinea pigs. A conjugate containing 2′(3′)-O-succinoyladenosine failed to elicit antibodies, whereas one containing laevulinic acid (O2′, 3′-adenosine-acetal) elicited antibodies in all animals injected. The affinity and specificity of binding of adenosine to three selected antisera were evaluated. Dissociation constants of 31-187 nM were observed. Displacement of adenosine binding to all antisera by adenine 5′-nucleotides, adenine, inosine and hypoxanthine required more than 1000-fold higher concentrations than of adenosine itself. Similar affinities for adenosine and 2′-deoxyadenosine were observed. By exploiting the high specificity of the antisera, a radioimmunoassay method was established that was capable of detecting down to 1 pmol of adenosine (20 nM) in unfractionated heart perfusates and cell extracts. The acetal-mediated coupling procedure is applicable to other biologically important cis-diols.

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