Cocaine/Benzoylecygonine Direct ELISA

Catalog Number SE120036

Storage Temperature 2–8 °C

TECHNICAL BULLETIN

Product Description The Cocaine/Benzoylecgonine Direct ELISA Kit avoids

Cocaine abuse is widespread and its prevalence may extraction of urine or blood samples for measurement.
1
be increasing in all social and age strata. The drug is It employs a benzoylecgonine directed antiserum. Due
1,2
generally inhaled or smoked. Several methods for to the proprietary method of orienting the antibody on
measurement of cocaine metabolites in urine exist.3-6 the polystyrene microplate much higher sensitivity is
Benzoylecgonine, a major metabolite, appears within achieved compared to passive adsorption. This allows
minutes in urine.3 Since the number and proportion of an extremely small sample size, reducing matrix
metabolites vary in subjects, results are expressed in effects, and interference with binding proteins(s) or
benzoylecgonine equivalents per ml. The Cocaine/ other macromolecules
Benzoylecgonine Direct ELISA Kit is a single incubation
assay providing results similar to those obtained by The Cocaine/Benzoylecgonine Direct ELISA Kit is a
existing methods.4-6 Native (unaltered) cocaine urine specific and sensitive in vitro test to detect the presence
concentration is far lower than that of its major of benzoylecgonine (BE) in forensic samples such as
metabolite benzoylecgonine. After intravenous whole blood, serum, plasma, and urine.
administration of 100 mg of cocaine, urine
concentrations ranged from 1.2–2.4 µg/ml compared Components
with concentrations ranging from 5–55 µg/ml for
benzoylecgonine.3 Cocaine was undetectable (at a Materials Provided 96 Tests
50 ng/ml cut-off) 12 hours after administration in Microwells 12 × 8 × 1
comparison with benzoylecgonine which persists hours BE-Conjugate 12 ml
to days after administration.7 It has been suggested Immunalysis Positive Reference
that a benzoylecgonine/cocaine ratio of less than 100 is 2 ml
Standard
indicative of use within the past 10 hours.7 Negative Standard 1 ml
TMB Substrate 12 ml
The Cocaine/Benzoylecgonine Direct ELISA Kit is Stop Reagent 11 ml
based upon the competitive binding to antibody of
enzyme labeled antigen and unlabeled antigen, in Reagents and Equipment Required but Not
proportion to their concentration in the reaction mixture. Provided.
A 10 µl aliquot of a diluted unknown specimen is • Distilled or deionized water
incubated with a 100 µl dilution of enzyme (Horseradish • Precision pipettes
peroxidase) labeled benzoylecgonine derivative in • Disposable pipette tips
microplate wells, coated with fixed amounts of oriented
• ELISA reader capable of reading absorbance at
high affinity purified polyclonal antibody. The wells are
450 nm
washed thoroughly and a chromogenic substrate
• Absorbent paper or paper towel
added. The color produced is stopped using a dilute
acid stop solution and the wells read at 450 nm. The • Graph paper
intensity of the color developed is inversely proportional
Precautions and Disclaimer
to the concentration of drug in the sample. The
technique is sensitive to 1 ng/ml. This product is for R&D use only, not for drug,
household, or other uses. Please consult the Safety
Data Sheet for information regarding hazards and safe
handling practices.
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Preparation Instructions 3. Add 10 µl of the diluted specimens into selected

Sample Preparation wells. It is recommended samples be run in
1. The Cocaine/Benzoylecgonine Direct ELISA Kit is duplicate.
to be used with human forensic samples, like whole 4. Add 100 µl of the BE-Conjugate to each well. Tap
blood, serum, and plasma. All possible applications the sides of the plate holder to ensure proper
of this assay have not been tested. Cutoff criteria mixing.
are important in deciding the sample dilution. 5. Incubate for 60 minutes at room temperature
2. Specimens to which sodium azide has been added (18–26 °C) preferably in the dark, after addition of
affect the assay. BE-Conjugate to the last well.
3. Urine samples should be stored at 2–8 °C until use. 6. Wash the wells 6 times with 350 µl of distilled water
Samples should be well mixed or vortexed before using either a suitable plate washer or wash bottle
assay. taking care not to cross contaminate wells. If testing
4. Repeated freezing and thawing should be avoided. samples containing abnormally high amounts of
Urine samples should be shipped refrigerated with hemoglobin (some postmortem samples), use
ice packs or equivalent. 10 mM Phosphate Buffered Saline, pH 7.0-7.4. This
will lower potential non-specific binding of
Storage/Stability hemoglobin to the well, thus lowering background
Store the kit at 2–8 °C. Keep microwells sealed in a dry color.
bag with desiccants. The reagents are stable until 7. Invert wells and vigorously slap dry on absorbent
expiration of the kit. Do not expose reagents to heat, paper to ensure all residual moisture is removed.
sun, or strong light This step is critical to ensure residual BE-Conjugate
does not skew results. If using an automated
Procedure system, ensure the final aspiration on the wash
Notes: The components in this kit are intended for use cycle aspirates from either side of the well.
as an integral unit. The components of different lots 8. Add 100 µl of TMB Substrate reagent to each well
should not be mixed. and tap sides of plate holder to ensure proper
mixing.
It is recommended diluted standards and diluted 9. Incubate for 30 minutes at room temperature,
specimens be run in duplicate. preferably in the dark.
10. Add 100 µl of Stop Solution to each well, to change
Optimal results will be obtained by strict adherence to the blue color to yellow.
this protocol. Accurate and precise pipetting, as well as 11. Measure the absorbance at a dual wavelength of
following the exact time and temperature requirements 450 nm and 650 nm.
prescribed are essential. Any deviation from this may 12. Wells should be read within 1 hour of yellow color
yield invalid data. development.
All reagents must be brought to room temperature Results
(18–26 °C) before use and gently mix. The following data represent a typical dose/response
curve.
1. Dilute forensic specimens, to the necessary range
with Phosphate Buffered Saline, pH 7.0. (Urine Benzoylecgonine (ng/ml) Absorbance
samples are normally diluted 1:10 for a 0 2.2
Benzoylecgonine cutoff of 300 ng/ml.) The dilution 10 0.52
factor and volume added can be adjusted based on
25 0.33
the laboratory’s cutoff.
50 0.27
2. Add 10 µl of appropriately diluted standards into
selected wells. It is recommended standards be run
The dose/response curve shown above should not be
in duplicate.
used in assay calculations. It is recommended that at
least one in-house positive quality control sample be
included with every assay run. A dose response curve
or a cutoff calibrator should be run with every plate.
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References
1. Urine Testing for Drugs of Abuse, National Institute 4. Diagnostic Products Corp.- Double Antibody
on Drug Abuse Research Monograph. 73, 95-97 COCAINE/BENZOYLECGONINE Assay.
(1986). 5. Roche Diagnostics. Abuscreen COCAINE/
2. Siegel, R.K., in: National Institute on Drug Abuse. BENZOYLECGONINE Radioimmunoassay.
Research Monograph Series 50. pp. 92-110 (1986). 6. Syva Corp. EMIT COCAINE/BENZOYLECGONINE
3. Kogan, M.J. et al., Anal. Chem., 49, 1965 (1977). Assay.
7. Ambre, J., J. Anal. Toxicol., 9, 241 (1985).

CH,SG,MAM 09/14-1

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