Polyclonal antibodies to agonist benzodiazepines

Abstract

Benzodiazepine-binding, immunoglobulin G class antibodies have been raised in three rabbits immunised with a conjugate of kenazepine coupled to keyhole limpet haemocyanin. The antibodies were assayed by [ 3H]flunitrazepam binding, followed by adsorption onto Staphylococcus aureus cells. Measurement of the rates of association and dissociation of [ 3H]flunitrazepam binding, together with saturation analysis of equilibrium binding, revealed varying degrees of heterogeneity in the affinity constants of the three rabbit antisera (equilibrium K D values 0.18 to 4.13 nM at 20–22°). Specificity of the antibodies was investigated by testing a wide variety of compounds (at concentrations of up to 10–100 (μm) for their ability to inhibit [ 3H]flunitrazepani binding. Only benzodiazepines known to act as agonists at their receptor sites in the central nervous system (CNS) caused an inhibition of binding. The rank orders of the IC 50 values of these drugs for inhibition of [ 3H]flunitrazepam binding to IgG from two out of the three rabbits correlated significantly with that previously published for displacement of CNS receptor binding. The agonist β-carboline derivative ZK 93423, the anxiolytic cyclopyrrolones suriclone and zopiclone and the purines inosine and hypoxanthine all failed to inhibit antibody binding, supporting previous suggestions that these drugs may bind at non-benzodiazepine recognition sites on the CNS receptor. The antibodies described are expected to provide useful reagents for raising anti-idiotypic antibodies directed against the CNS receptor and for the identification and purification of possible endogenous benzodiazepine receptor agonists in the CNS.