Characterisation of antibody models of the ryanodine receptor for use in high-throughput screening

The syntheses of seven novel synthetic analogues of the naturally occurring insecticide ryanodine are described. These, and other synthetic and naturally occurring analogues, have been used to characterize the selectivity of a monoclonal antibody which has been produced by immunization with 9-hydroxy-21-(4-azidobenzyloxy)-9-epiryanodine photo-conjugated to keyhole limpet haemocyanin. The antibody binds [³H]ryanodine with a dissociation constant of 0.37 nM. The specificity of this antibody in terms of its ability to recognise 11 natural and synthetic analogues of ryanodine has been determined by [³H]ryanodine displacement and shown to be similar (for a partially overlapping set of analogues) to that determined earlier for a rabbit polyclonal antibody (Kahl, S. D., et al., Anal. Biochem., 218 (1994) 55-62). The selectivity of the antibodies is shown to be related to that of the sarcoplasmic reticulum Ca²⁺ release channelfrom rabbit skeletal muscle, both for this set of ryanodine analogues and for three structurally dissimilar, low-molecular-weight compounds identified by high-throughput screening. The advantages of these antibody models of the Ca²⁺ release channel for screening are illustrated by their superior performance in a homogeneous bindingassay. [ABSTRACT FROM AUTHOR]