J. Biochem, 1974, Vol. 76, No. 6 1293-1302
© 1974 Japanese Biochemical Society
research-article |
Characterization of Specific Antibodies to Phenobarbital
Department of Biochemical Toxicology, School of Pharmaceutical Science, Showa University Shinagawa-ku, Tokyo 142
The specificities of antibodies to phenobarbital were examined by radioimmunoassay.
The antibodies were highly specific for the barbituric ring and required a 6-membered ring with an intact barbiturate urea moiety for large binding affinity. 2-Deoxyphenobarbital (primidon) showed no binding ability and barbiturates with substituents on the nitrogen atoms of the barbituric ring were found to have extremely low binding affinities. In contrast, barbiturates without N substituents possessed a binding affinity for the antibodies which depended upon the analogy of the structure to that of phenobarbital. Among them, cyclobarbital and heptabarbital, which have structures similar to that of phenobarbital, had high affinities. However mephobarbital (N-methylphenobarbital) had an affinity higher than that of barbital; its structure is the same as that of phenobarbital except for the N-methyl group.
The antibodies apparently did not discriminate the major metabolite of phenobarbital, p-hydroxyphenobarbital, which has the hydroxy group on the binding site of phenobarbital to carrier protein. From the findings with p-azophenobarbital-N-acetyltyrosine, the antigenic determinant of the antigen was assumed to include parts of the carrier protein.