Benzodiazepines Assay for Urine
IVD
Store at 2 - 8ºC.
INTENDED USE
The Benzodiazepine Enzyme Immunoassay (EIA) is a homogeneous enzyme
immunoassay system intended for use in the qualitative and semi-quantitative
analysis of benzodiazepines in human urine.
The assay provides only a preliminary analytical result. A more
specific alternative chemical method must be used in order to obtain
a confirmed analytical result. Gas Chromatography/mass
spectrometry (GC/MS) is the preferred confirmatory method (1, 2).
Clinical consideration and professional judgement should be
exercised to any result, in order to establish the appropriate
treatment or therapy.
PRINCIPLE OF THE METHOD
The benzodiazepine assay is a homogeneous enzyme immunoassay (3) with
ready-to-use liquid reagent. The assay is based on competition between drug
in the sample and drug labelled with the enzyme glucose-6-phosphate
dehydrogenase (G6PDH) for a fixed amount of antibody in the reagent.
Enzyme activity decreases upon binding to the antibody, and the drug
concentration in the sample is measured in terms of enzyme activity.
In the absence of drug in the sample, benzodiazepine-labelled G6PDH
conjugate is bound to antibody, and the enzyme activity is inhibited. On the
other hand, when free drug is present in the sample, antibody would bind to
free drug; the unbound benzodiazepine-labelled G6PDH then exhibits its
maximal enzyme activity. Active enzyme converts nicotinamide adenine
dinucleotide (NAD) to NADH, resulting in an absorbance change that can be
measured spectrophotometrically at 340 nm.
SIGNIFICANCE OF THE TEST
The benzodiazepine family has a diverse chemical structure which gives rise
to a range of physio-chemical and pharmacological properties. They form a
large group and are used as sedatives and in the treatment of anxiety. It is
estimated that over 50 benzodiazepine drugs are in common use world-wide
with approximately a dozen marketed within a give country. Benzodiazepines
are strongly associated with opioid users (4).
They are usually taken orally (although intravenous and intramuscular routes
are also used) and as would be expected from such a diverse group, the
pharmacological effects are broad and are also related with the absorption
rate. In general, they are extensively metabolised by dealkylation,
hydroxylation, oxidation and glucuronidation in the liver.
Some metabolites have biological activity; for example nordiazepam from
diazepam and oxazepam from temazepam. Detection of benzodiazepines and
their metabolites in urine can be used as an indication for use of
benzodiazepines.
REAGENTS
Antibody/Substrate Reagent (R1): Contains mouse monoclonal
antibodies benzodiazepines, glucose-6-phosphate (G6P), nicotinamide
adenine dinucleotide (NAD), stabilizers, and sodium azide as preservative.
Enzyme-drug Conjugate Reagent (R2): Contains benzodiazepine-labelled
glucose-6-phosphate dehydrogenase (G6PDH) in buffer with sodium azide as
preservative.
Avoid prolonged exposures of the reagent at temperatures higher
than 25°C.
CALIBRATORS AND CONTROLS
Negative Human Urine (Level 0): Contains negative human urine with
sodium azide as preservative (Ref.: 933010 and 933015).
INTERPRETATION
For qualitative determinations, the cut-off calibrator (200 or 300ng/mL) of
benzodiazepine is used as a reference for distinguishing positive from
negative samples. A sample with a change in absorbance ( mA/min) equal
to, or greater than, that obtained with the cut-off calibrator is considered
positive. A sample with a change in absorbance value lower than that
obtained with the cut-off calibrator is considered negative.
For semi-quantitative determinations, a calibration curve with multiple
calibrators is required. The concentration of benzodiazepines in the sample
may then be estimated from the calibration curve.
QUALITY CONTROL
Good laboratory practices recommend the use of control specimens to ensure
proper assay performance.
The calibration curve can be validated with the Control levels 150 and 250
ng/mL (ref. 936081) or levels 225 and 375 ng/mL (ref. 936082), or with
commercial controls.
LIMITATIONS
1.A positive result from the assay indicates only the presence of
benzodiazepines.
2.Positive results should be confirmed by other affirmative, analytical
methods (e.g. chromatography), and preferably GC/MS.
3.The test is designed for use with human urine only.
