Radioimmunoassay for the calcium release channel agonist ryanodine

A novel photo-activatable derivative of ryanodine, 9-hydroxy-21-(4-azidobenzoyloxy)-9-epiryanodine, has been synthesized and conjugated to keyhole limpet hemocyanin for the production of antibodies with high affinity and specificity to ryanodine. The anti-ryanodine antibodies reacted specifically on immunoblots with the azido-ryanodine compound covalently conjugated to bovine serum albumin. A radioimmunoassay specific for ryanodine was developed using the anti-ryanodine antibodies, and a dissociation constant for ryanodine of 1 nM was determined. Half-maximal inhibition constants (IC50) for various ryanodine derivatives were found to range between 3.2 and 200 nM. These IC50 values correlated very well with the IC50 values obtained for the compounds binding to the skeletal muscle membrane receptor. These antibodies should be useful for the characterization of the ryanodine binding site on the sarcoplasmic reticulum Ca2+ release channel.