Production of a High Affinity Antiserum to Benzodiazepines

Abstract

Antibodies specific for benzodiazepines were raised in rabbits by immunization with a conjugate of a benzodiazepine derivative, Ro 7-1986/1, with bovine serum albumin. The presence of anti-Ro 7-1986/1 antibodies in the sera was demonstrated by a radioimmunoassay using the radioligand [3H]flunitrazepam ([3h]FNZ). The antibodies displayed a high-affinity for [3H]FNZ (KD = 0.073 +/- 0.003 nM) and and cross-reacted with a broad spectrum of benzodiazepine derivatives. Benzodiazepine levels in samples of sera and urine of benzodiazepine-treated humans were determined. Due to the high sensitivity of the assay only minute volumes (microliter quantities) of body fluids are employed and, therefore, no extraction of the drugs is required. Nitrazepam and diazepam levels as low as 20 picograms can be easily observed. Intoxicating levels of benzodiazepines can be detected by a single measurement in less than 10 min. This radioimmunoassay is advantageous for pharmacokinetic studies, toxicological examinations and forensic medicine due to its high sensitivity, wide-range specificity and technical simplicity.

Monoclonal Antibodies to Benzodiazepines

“…The mAbs allow us (a) to study the mAb-benzodiazepine binding as a bimolecular reaction and (b) to maintain for many years (theoretically forever) the hybridoma lines that will always produce the same antibody, therefore avoiding the variability in quality and quantity among the conventional antisera obtained not only from different animals, but also from the different bleedings of the same animal. The specificities and affinities of the mAbs for benzodiazepines ( Table 3) are different from those of several conventional antisera reported in the literature (Budd, 1981;Dixon, 1982;Sherman-Gold et al, 1983). The affinities of [3H]FNZ for both the mAbs and the brain benzodiazepine receptors are similar.…”

“…We have generated four mAbs with high affinities for [3H]FNZ and other benzodiazepine receptor agonists. Conventional antisera to benzodiazepines have been generated by others (Peskar and Spector, 1973; Early et al, 1979;Budd, 1981;Dixon, 1982;Sherman-Gold et al, 1983). Nevertheless, our antibodies have many advantages derived from the fact that they are homogeneous molecules (all the mAbs secreted by a hybridoma line are identical) that can be supplied in unlimited quantities by immortal hybridoma lines.…”

“…Benzodiazepines are widely prescribed drugs that have anxiolytic, anticonvulsant, muscle relaxant, and hypnotic properties (Costa and Guidotti, 1979;Tallman et al, 1980). Conventional antisera to various benzodiazepines have been produced by others (Peskar and Spector, 1973;Early et al, 1979;Budd, 1981;Dixon, 1982;Sherman-Gold et al, 1983), and such antisera have been used for the immunoassay determination of these drugs in blood, saliva, and urine. The main advantage of the immunoassays is that they are very simple procedures in comparison with the alternative benzodiazepine assay methods, such as electron-capture gas chromatography, spectrophotofluorometry, differential pulse poiarography, etc.…”