New fluorescence polarization immunoassays for analysis of barbiturates and benzodiazepines in serum and urine: performance characteristics.

The performance of the new fluorescence polarization immunoassay reagents Cassette COBAS INTEGRA Serum Benzodiazepines assay (SBENZ) and Cassette Serum Barbiturates assay (SBARB) was evaluated as compared to other immunoassays (Abbott TDx Serum Benzodiazepines, Abbott TDx Urine Benzodiazepines, Behring EMIT Serum Benzodiazepines, Abbott ADx Serum Barbiturates, Behring EMIT Serum Barbiturates, and the COBAS INTEGRA Barbiturates (BARB) urine assay) and gas chromatography-mass spectrometry (GC-MS). Recoveries of nordiazepam and secobarbital using the SBENZ and SBARB assays, respectively, were equivalent for serum, plasma, and urine. Cross-reactivities of structurally related benzodiazepines, barbiturates, and their metabolites were very similar in serum and urine for the SBENZ and SBARB assays. Precision was within 5.4% for SBENZ serum and within 11% from 10 to 100 ng/mL for urine. Precision was within 5% for SBARB serum and within 7% from 136 to 277 ng/mL for the urine application. The standard curves for SBENZ and SBARB were stable for at least 16 weeks with the reagents stored open on the COBAS INTEGRA analyzer. Clinical comparison of the SBENZ serum assay indicated an increased pickup rate, as confirmed by GC-MS, compared to TDx and EMIT. The diagnostic sensitivities of the SBENZ serum application, TDx, and EMIT versus GC-MS were 100%, 89%, and 36%, respectively. The diagnostic specificities were 71%, 79%, and 100%, respectively. The diagnostic sensitivities of the SBENZ urine application and TDx versus GC-MS were 100% and the diagnostic specificities were 88%. The increased positive pick-up of the SBENZ assay compared to the other immunoassays is most probably due to the difference in the limit of detection (LOD) and the increased cross-reactivity for the low-dose benzodiazepines. Clinical comparison of the SBARB serum assay indicated an increased positive pick-up rate, as confirmed by GC-MS. The diagnostic sensitivities of the SBARB serum application, ADx, and EMIT versus GC-MS were 96%, 65%, and 35%, respectively. The diagnostic specificities were all 100%. The diagnostic sensitivities for the SBARB urine application and BARB versus GC-MS were all 100%, and the diagnostic specificities were all 91%. The SBENZ and SBARB kits demonstrated increased sensitivity for the detection of benzodiazepines and barbiturates in both serum and urine compared to the other immunoassays.

Synthesis of conjugates for a barbiturate screening assay.

Novel derivatives of barbiturates functionalized with free carboxylic acids were designed and synthesized. Coupling of 5-cyclopentyl-5-carboxycrotylbarbituric acid via its active ester to an aminofluorescein derivative produced a fluorescent tracer. Conjugation of the 5-cyclopentenyl-5-carboxyethylbarbituric acid via its mixed anhydride to thyroglobulin allowed for subsequent development of a polyclonal antibody which was evaluated for binding in a fluorescence polarization immunoassay format with various barbiturates. The binding studies showed good cross-reactivity of a variety of barbiturates containing both aromatic and aliphatic 5-substituents with the tested antisera. The relationship between the immunogen architecture, the chemical structure of the binding analytes, and the characteristics of the antisera is also presented.

[Immunologic studies of barbiturates--the effect of structural characteristics on cross-reactivity]. / Immunologische Untersuchungen auf Barbiturate--Der Einfluss von Strukturmerkmalen auf die Kreuzreaktivität.

Immunologic assay methods are widely used in chemico-toxicological analysis. Often test kits that are created for the detection of entire drug groups, e.g. barbiturates, are put into action. Within a drug-group the extent of the reaction with the test antiserum, the cross-reactivity, varies considerably. The influence of molecular structures on the cross-reactivity is examined for the ADX-test kit for the detection of barbiturates from urine. In order to obtain a result equal to or exceeding the threshold value (200 ng/ml secobarbital-equivalents) high barbiturate concentrations in urine are required in some cases.

Drugs as allergens: the molecular basis of IgE binding to thiopentone.

Using an immunoassay developed for the detection of thiopentone-reactive IgE antibodies, the combining site specificities of such antibodies found in sera of patients who experienced life-threatening anaphylactic reactions to the drug were studied. The antibody combining sites from one patient were complementary to the region of the thiopentone molecule containing a thio group at position 2 of the barbiturate ring. The allergenic determinant recognized by IgE antibodies from another patient encompassed a secondary pentyl group and an ethyl group attached to position 5 on the other side of the barbiturate ring. Thus, it is already clear that there is more than one allergenic determinant on the thiopentone molecule with the capacity to provoke IgE formation and drug-induced allergic reactions.

Pharmacologic response to pentobarbital in actively immunized mice.

Mice were placed on an immunization schedule known to result in the production of antibodies directed against a variety of barbiturates (antibody binding capacity = 2.71 pmole 3H-phenobarbital bound/ml undiluted serum). The pharmacologic response to barbiturate in these actively immunized mice and suitably treated controls was investigates using rotarod apparatus for monitoring CNS depression. It was found that the response to pentobarbital in actively immunized mice was decreased, as reflected by an increase in the time the mice remained on the rotarod and a shift of the dose--response curve to the right. The decreased pharmacologic response to pentobarbital was not the result of changes in levels of the hepatic drug metabolism components. Furthermore, the alteration of pharmacologic response in actively immunized mice was selective for barbiturate and did not modify the ataxia produced by administration of another depressant agent, ethanol.