Haloperidol binding to monoclonal antibodies: conformational analysis and relationships to D-2 receptor binding

A library of 22 monoclonal antibodies to the D-2 dopaminergic receptor antagonist haloperidol has been developed by immunizing BALB/c mice with two conformationally distinct immunogens. The two immunogens were prepared by coupling haloperidol to bovine serum albumin through the tertiary alcohol at the 4-position of the piperidine ring with a succinic acid linkage and by coupling to bovine serum albumin through the ketone group of the butyrophenone with a carboxymethyl oxime linkage. Seventeen monoclonal antibodies displayed specific, saturable, high affinity binding of [3H]haloperidol which could be inhibited by a variety of neuroleptic drugs. Three monoclonal antibodies raised against the succinic acid conjugate and two monoclonal antibodies raised against the oxime conjugate were selected for detailed analysis of the molecular characteristics of binding specificity and for relationships to bovine striatal D-2 dopaminergic receptor binding. The monoclonal antibody with highest affinity for haloperidol, 185(2)-1 (raised against the succinic acid conjugate and herein referred to as mAb A), had a Kd of 3.3 (+/- 0.06) nM and primarily recognized chemical determinants in the butyrophenone ring (ring 1) and side chain. Inhibition of [3H]haloperidol binding to mAb A by 16 unlabeled haloperidol analogs displayed a good correlation [r = 0.82, n = 16, m = 1.06(+/- 0.38)] with D-2 receptor binding affinity, suggesting that the parts of the D-2 receptor combining site which recognize butyrophenone antagonists may have molecular characteristics which are similar to those of the monoclonal antibody. Other dopaminergic ligands such as dopamine and the D-1 antagonist SCH-23390 were not recognized by monoclonal antibodies raised against the succinic acid conjugate. Monoclonal antibodies raised against the oxime conjugate such as 258(2)-1 (herein referred to as mAb D) primarily recognized chemical determinants in ring 2 and the tertiary amino group of the piperidine ring. Although the D-2 receptor and mAb D both prefer electron-withdrawing substituents in the para position of ring 2, the antibody was more sensitive than the receptor to changes and displayed affinities that were much lower for substituents attached to the p-chlorophenyl ring (ring 2), which were electron donating. In addition, dopamine was able to completely displace 4 nM [3H]haloperidol from mAb D at a concentration of 6 mM.