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Smoke Check
Test
One-Step Cotinine Test
A visual one-step immunoassay for the qualitative detection of cotinine
in human urine.
For determination of smoking status only. Not Intended For Medical
Diagnostic Use.
Order Now! Value pricing as low as $3.60
per test! 
INTENDED USE
The Smoke Check Test is a lateral flow, one-step immunoassay for
the qualitative detection of cotinine, the major metabolite of nicotine
in human urine, at a cut-off concentration of 200 ng/ml. This product
is used to obtain a visual, qualitative result and is intended for
the determination of smoking status only. This assay provides only
a preliminary analytical test result. A more specific alternative
chemical method such as high performance liquid chromatography (HPLC)
or gas chromatography/mass spectrometry (GC/MS) must be used in
order to obtain a confirmed analytical result. Clinical consideration
and professional judgment should be applied to all test results,
particularly when preliminary positive results are indicated.
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SUMMARY
Tobacco smoking results in the absorption of nicotine through the
lung and buccal/nasal epithelium, after which nicotine is metabolized
into about 20 metabolites excreted in urine. Cotinine, a major metabolite,
accumulates in the body with regular smoking. It is reported that
cotinine is stable in body fluids and has a relatively long half
life of approximately 17 hours. Therefore, the detection of cotinine
is less dependent on the time of sampling than that of nicotine
and other metabolites. Cotinine has been widely used as a biomarker
of tobacco exposure. Methods of analysis for cotinine in biological
fluids include gas chromatography, gas chromatography-mass spectrometry,
HPLC, HPLC-mass spectrometry, EIA and RIA. These methods usually
require special equipment and complicated operation procedures.
The Smoke Check Test is a one step immunoassay that is used for
the qualitative detection of cotinine in human urine. It is based
on the principle of highly specific immunochemical reactions of
antigens and antibodies. It is a simple and convenient test for
the rapid qualitative detection of cotinine in human urine at 200
ng/ml cut-off concentration.
Note: A very faint line in the test region indicates that the cotinine
concentration in urine is near the cut-off level for the test. These
samples should be re-tested or confirmed with a more specific method
before a positive result is determined.
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TEST PRINCIPLE
The Smoke Check Test applies the principle of a competitive immunoassay.
The test device contains a membrane strip that is precoated with
cotinine antigen at the test line region. The cotinine antibody
gold conjugate pad is placed at the end of the membrane. In cotinine-free
urine, the colored antibody-colloidal gold conjugate and urine moves
chromatographically by capillary action across the membrane. This
solution migrates to the test line containing cotinine antigen and
forms a visible line as the antibody complexes with the antigen.
The formation of a visible precipitant in the test zone indicates
a negative result (non-smoker). When cotinine is present in urine,
it competes with cotinine on the test band region for the limited
antibody sites. When a sufficient concentration of cotinine is present
in urine, it will fill the limited antibody binding sites. This
will prevent attachment of the colored antibody-colloidal gold conjugate
at the test line region. Therefore, absence of the color band on
the test region indicates a positive result (smoker). A different
antigen/antibody reaction is added to the membrane strip at the
control region (C) to indicate that the test has been performed
properly. This control line should always appear, regardless of
the cotinine status in the urine. This means that negative urine
will have two pink colored bands, and positive urine will have only
one pink colored band. The pink colored control band serves as an
indicator that 1) A sufficient volume of urine has been added, and
2) that proper flow was obtained.
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STORAGE AND STABILITY
The test kit should be stored refrigerated or at room temperature
2-307C (36-867F). Each device should remain in its sealed pouch
for the duration of the shelf-life.
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PRECAUTIONS
• Urine specimens may be potentially infectious. Proper handling
and disposal methods should be established.
• Avoid cross-contamination of urine samples by using a new
specimen collection container and specimen pipette for each urine
sample.
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REAGENTS AND MATERIALS SUPPLIED
• Individually wrapped test devices which include one disposable
pipette each.
• One instruction sheet.
MATERIAL REQUIRED BUT NOT PROVIDED
• Specimen collection container.
• Timer.
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SPECIMEN COLLECTION AND HANDLING
The urine specimen must be collected in a clean dry container,
either plastic or glass. Fresh urine does not require any special
handling or pretreatment. Test should be performed soon after the
urine specimen is collected, preferably during the same day. The
specimen may be refrigerated at 2-87C for 3 days or frozen at -207C
for a longer period of time. Specimens that have been refrigerated
must be equilibrated to room temperature prior to testing. Specimens
previously frozen must be thawed, equilibrated to room temperature,
and mixed thoroughly prior to testing.
Note: Urine specimens and all materials coming in contact with
them should be handled and disposed of as if capable of transmitting
infection. Avoid contact with skin by wearing gloves and proper
laboratory attire.
Improve reliability of your test results by testing the specimen
with the Urine ID Adulteration Stick. The Urine ID Stick tests the
specimen against tampering any of various substances it may have
been diluted with. 
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 TEST
PROCEDURE
Review "Specimen Collection" instructions. If refrigerated,
test device, patient samples, and controls should be brought to
room temperature (20-307C) prior to testing. Do not open pouches
until ready to perform the assay.
1. Remove the test device from its protective pouch (bring the
device to room temperature before opening the pouch to avoid condensation
of moisture on the membrane). Label the device with patient or control
identification.
2. Draw the urine sample to the line marked on the pipette (approximately
0.2 ml). Dispense the entire contents into the sample well. Use
a separate pipette and device for each sample or control.
3. Read result between 3 to 8 minutes after the addition of samples.
Do not read result after 8 minutes.
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INTERPRETATION OF RESULTS
Negative:
Both the test line (T) and the control line (C) should appear in
the viewing window. The control line (C) indicates proper performance
of the device. The test line intensity may be weaker or stronger
than that of the control line.
Positive:
Only one colored line appears in the control line region (C). No
colored line appears in the test line region (T).
Invalid:
No colored line appears in the control region. Under no circumstances
should a positive result be identified unless the control line (C)
appears in the viewing area. If the control line (C) does not appear,
the test result is invalid and the assay should be repeated.
C. Precision
The precision of the Smoke Check Test was determined by conducting
the test with spiked controls. The control at 100 ng/ml gave a negative
result. The control at 400 ng/ml gave a positive result.
D. Specificity
The specificity for the Smoke Check Test was tested by adding various
drugs, drug metabolites, and other compounds that are likely to
be present in urine. All compounds were prepared in cotinine-free
normal human urine.
The following structurally related compounds produced positive
results when tested at levels equal to or greater than the concentrations
listed below.
Compound Concentration
(-)-Nicotine 350 ug/ml
The following compounds were found not to cross-react when tested
at concentrations up to 100 µg/ml.
Acetaminophen DL-Homatropine
Acetylsalicylic Acid Hydrocodone
Albumin Hydromorphone
Amitriptyline (+/-)-Isoproterenol
D-Amphetamine Lidocaine
Ampicillin Maprotiline
Aspartame Methadone
Aspirin Meperidine
Atropine Methadol
Benzocaine Methamphetamine
Benzoylecgonine Methapyrilene
(+)-Brompheniramine Methaqualone
Caffeine Methylphenidate
Chloroquine (+/-)3,4-MDMA
(+)-Chlorpheniramine Morphine
(+/-)-Chlorpheniramine Morphine-3-ß-d-glucuronide
Chlorprothixene (1S,2S)-(-)-N-Methyl-Ephedrine
Codeine Naloxone
Creatine Naltrexone
r-Cyclodextrin ß-Naphthaleneacetic acid
Cyclobenzaprine (+)-Naproxen
(-)-Deoxyephedrine Nortriptyline
Dextromethorphan Nicotinic Acid
4-Dimethylaminoantipyrine Oxalic Acid
5,5-Diphenylhydantoin Penicillin-G
Diazepam Pentobarbital
Dopamine Perphenazine
Doxylamine Pheniramine
Ecgonine Methyl Ester Phenobarbital
EDDP Phenothiazine
(-)-Ephedrine L-Phenylephrine
(+)-Ephedrine a-Phenylethylamine
(+/-)-Ephedrine Phentermine
(+/-)-Epinephrine (+/-)-Phenylpropanolamine
Erythromycin Primidone
Furosemide Procaine
Glucose Promethazine
Guaiacol Glyceryl Ether d-Propoxyphene
Hemoglobin Secobarbital
Sodium Chloride D(+)Trehalose
Tenocyclidine Trifluoperazine
.9-Tetrahydrocannabinol Tyramine
Theophylline Triprolidine Hydrochloride
Thioridazine Vitamin C
Trimethobenzamide
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QUALITY CONTROL
Good laboratory practice recommends the use of control materials
to ensure proper kit performance. Before using a new kit with patient
specimens, positive and negative controls should be tested. Quality
control specimens are available from commercial sources. When testing
the positive and negative controls, use the same assay procedure
as with a urine specimen.
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LIMITATIONS
• The assay is designed for use with human urine only.
• A positive result indicates only that the presence of cotinine
is above the cut off concentration. It does not indicate or measure
level of consumption.
• There is a possibility that technical or procedural errors
as well as other substances or factors not listed may interfere
with the test and cause false results. See SPECIFICITY for lists
of substances that will produce positive results, or that do not
interfere with test performance.
• If it is suspected that the samples have been mislabeled
or deteriorated, a new specimen should be collected and the test
should be repeated.
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PERFORMANCE CHARACTERISTICS
A. Accuracy
The accuracy of the Smoke Check Test was evaluated in comparison
to a commercially available immunoassay at a cut off concentration
of 200 ng/ml for cotinine. One hundred twenty samples (120), collected
from presumed non-smoker volunteers, were tested by both methods
with 100% agreement. In a separate study, fifty (50) urine specimens
with cotinine concentrations ranging from 300 ng/ml to 2,000 ng/ml
(as determined by a commercially available immunoassay) were tested
using the Smoke Check Test. All fifty (50) specimens were determined
positive by the Smoke Check Test, representing 100 % agreement with
the commercially available immunoassay.
B. Reproducibility
The reproducibility of the Smoke Check Test was evaluated at four
different sites using blind controls. Of the sixty (60) samples
with cotinine concentrations of 100 ng/ml, all were determined negative.
Of the sixty (60) samples with cotinine concentrations of 400 ng/ml
of cotinine, all were determined positive.
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